Cell junction PDZ protein

ABSTRACT

The invention provides a human cell junction PDZ protein (CJPDZ) and polynucleotides which identify and encode CJPDZ. The invention also provides expression vectors, host cells, antibodies, agonists, and antagonists. The invention also provides methods for diagnosing, treating, or preventing disorders associated with expression of CJPDZ.

[0001] This application is a divisional application of U.S. applicationSer. No. 09/370,102 filed on Aug. 6, 1999, which is a divisionalapplication of U.S. application Ser. No. 09/151,611 filed on Sep. 11,1998, issued on Sep. 28, 1999, as U.S. Pat. No. 5,958,731, entitled CELLJUNCTION PDZ PROTEIN, the contents all of which are hereby incorporatedby reference.

FIELD OF THE INVENTION

[0002] This invention relates to nucleic acid and amino acid sequencesof a cell junction PDZ protein and to the use of these sequences in thediagnosis, treatment, and prevention of cancer, neurological disorders,and developmental disorders.

BACKGROUND OF THE INVENTION

[0003] Cells communicate with and respond to their environment byreceiving and processing extracellular signals. These signals take theform of growth factors, hormones, cytokines, and peptides which bind toand activate specific plasma membrane receptors. The activated receptorstrigger intracellular signal transduction pathways which culminate in awide range of cellular responses affecting gene expression, proteinsecretion, cell cycle progression, and cell differentiation. Initialevents in signal transduction require the proximity of intracellularsignaling proteins to the cytosolic domains of activated plasma membranereceptors. These intracellular membrane-associated signaling proteinscouple the activated receptor to downstream second messenger systems andplay a key role in the regulation and coordination of complex,multiprotein signal transduction pathways. Recently, a conserved proteindomain called the PDZ domain has been identified in variousmembrane-associated signaling proteins. This domain has been implicatedin receptor and ion channel clustering and in the targeting ofmultiprotein signaling complexes to specialized functional regions ofthe cytosolic face of the plasma membrane. (For review of PDZdomain-containing proteins, see Ponting, C. P. et al. (1997) Bioessays19:469-479.)

[0004] PDZ domains were named for three proteins in which this domainwas initially discovered. These proteins include PSD-95 (postsynapticdensity 95), Dlg (Drosophila lethal(1)discs large-1), and ZO-1 (zonulaoccludens-1). These proteins play important roles in neuronal synaptictransmission, tumor suppression, and cell junction formation,respectively. Since the discovery of these proteins, over sixtyadditional PDZ-containing proteins have been identified in diverseprokaryotic and eukaryotic organisms. A large proportion of PDZ domainsare found in the eukaryotic MAGUK (membrane-associated guanylate kinase)protein family, members of which bind to the intracellular domains ofreceptors and channels. However, PDZ domains are also found in diversemembrane-localized proteins such as protein tyrosine phosphatases,serine/threonine kinases, G-protein cofactors, and synapse-associatedproteins such as syntrophins and neuronal nitric oxide synthase (nNOS).Generally, about one to three PDZ domains are found in a given protein,although up to nine PDZ domains have been identified in a singleprotein.

[0005] X-ray crystallography has shown that PDZ domains are generallycompact globular structures containing about 80 to 100 amino acids whichform six P-strands and two a-helices. PDZ domains tend to be rich inglycine residues which introduce turns in the polypeptide chain andpromote compaction and stability of the folded polypeptide. Inparticular, a glycine-aspartic acid (GD) dipeptide and an asparagineresidue which occurs six residues thereafter are highly conserved innearly all PDZ domains and are important for domain structure. PDZdomains bind to a tripeptide motif containing valine and serine orthreonine. Most ligands which bind PDZ domains contain this motif,although some ligands lack this motif or contain conservativesubstitutions therein.

[0006] Members of the MAGUK protein family play an important role in theclustering of neurotransmitter receptors and channels, and thisclustering is essential for neuron function (Ponting, supra). In thepresence of a neurotransmitter, the PDZ domains of PSD-95, PSD-93,SAP-97 (synapse-associated protein 97), SAP-102, and chapsyn 110 bind tothe cytosolic C-termini of N-methyl-D-aspartate (NMDA) neurotransmitterreceptors and Shaker-type potassium channels, causing them to cluster.In addition, interaction of the PDZ domain of PSD-95 with that of nNOSanchors the latter in proximity to the NMDA receptors.

[0007] In the nematode Caenorhabditis elegans, the PDZ-containingprotein, LIN-7, is required during development for the specification ofcells which give rise to the vulva (Simske, J. S. et al. (1996) Cell85:195-204). These precursor cells form an epithelium in the sexuallyimmature nematode. A diffusible epidermal growth factor (EGF)-likesignal is secreted in the vicinity of the epithelium. Cells insufficiently close proximity to the signal respond by activating anEGF-like receptor tyrosine kinase/Ras-mediated signal transductionpathway. Responding cells subsequently give rise to generations ofprogeny cells which differentiate to form the vulva. The EGF-likereceptor (EGFR) is localized to epithelial cell junctions, and thislocalization is essential for signal transduction and vulva formation.The LIN-7 gene is required for EGFR localization and encodes a 297 aminoacid protein (SEQ ID NO: 3) which contains a PDZ domain from amino acid199 to amino acid 280.

[0008] PDZ-containing proteins are likely involved in disordersassociated with defective cell signaling (Ponting, supra). As discussedabove, PDZ domains have been shown to play important roles indevelopment, and in fact, the gene encoding the PDZ-containing protein,LIM kinase 1, is deleted in patients with Williams syndrome, a complexdevelopmental disorder. PDZ-containing proteins have also beenimplicated in oncogenesis. For example, mutations in Drosophila Dlgcauses neoplastic transformation of epithelial cells, and N-terminallytruncated forms of the PDZ-containing protein, Tiam 1, are highlytumorigenic in nude mice. In addition, mutations that block clusteringof neuronal receptors and channels cause perinatal lethality in mice,suggesting that the clustering function of neuronal MAGUK proteins iscritical.

[0009] The discovery of a new cell junction PDZ protein and thepolynucleotides encoding it satisfies a need in the art by providing newcompositions which are useful in the diagnosis, prevention, andtreatment of cancer, neurological disorders, and developmentaldisorders.

SUMMARY OF THE INVENTION

[0010] The invention is based on the discovery of a new human celljunction PDZ protein (CJPDZ), the polynucleotides encoding CJPDZ, andthe use of these compositions for the diagnosis, treatment, orprevention of cancer, neurological disorders, and developmentaldisorders.

[0011] The invention features a substantially purified polypeptidecomprising the amino acid sequence of SEQ ID NO: 1 or a fragment of SEQID NO:1.

[0012] The invention further provides a substantially purified varianthaving at least 90% amino acid sequence identity to the amino acidsequence of SEQ ID NO: 1 or a fragment of SEQ ID NO: 1. The inventionalso provides an isolated and purified polynucleotide encoding thepolypeptide comprising the amino acid sequence of SEQ ID NO: 1 or afragment of SEQ ID NO: 1. The invention also includes an isolated andpurified polynucleotide variant having at least 90% polynucleotidesequence identity to the polynucleotide encoding the polypeptidecomprising the amino acid sequence of SEQ ID NO: 1 or a fragment of SEQID NO: 1.

[0013] The invention further provides an isolated and purifiedpolynucleotide which hybridizes under stringent conditions to thepolynucleotide encoding the polypeptide comprising the amino acidsequence of SEQ ID NO: 1 or a fragment of SEQ ID NO: 1, as well as anisolated and purified polynucleotide having a sequence which iscomplementary to the polynucleotide encoding the polypeptide comprisingthe amino acid sequence of SEQ ID NO: 1 or a fragment of SEQ ID NO: 1.

[0014] The invention also provides an isolated and purifiedpolynucleotide comprising the polynucleotide sequence of SEQ ID NO:2 ora fragment of SEQ ID NO:2, and an isolated and purified polynucleotidevariant having at least 90% polynucleotide sequence identity to thepolynucleotide comprising the polynucleotide sequence of SEQ ID NO:2 ora fragment of SEQ ID NO:2. The invention also provides an isolated andpurified polynucleotide having a sequence complementary to thepolynucleotide comprising the polynucleotide sequence of SEQ ID NO:2 ora fragment of SEQ ID NO:2.

[0015] The invention also provides a method for detecting apolynucleotide in a sample containing nucleic acids, the methodcomprising the steps of (a) hybridizing the complement of thepolynucleotide sequence to at least one of the polynucleotides of thesample, thereby forming a hybridization complex; and (b) detecting thehybridization complex, wherein the presence of the hybridization complexcorrelates with the presence of a polynucleotide in the sample. In oneaspect, the method further comprises amplifying the polynucleotide priorto hybridization.

[0016] The invention further provides an expression vector containing atleast a fragment of the polynucleotide encoding the polypeptidecomprising the sequence of SEQ ID NO: 1 or a fragment of SEQ ID NO: 1.In another aspect, the expression vector is contained within a hostcell.

[0017] The invention also provides a method for producing a polypeptide,the method comprising the steps of: (a) culturing the host cellcontaining an expression vector containing at least a fragment of apolynucleotide under conditions suitable for the expression of thepolypeptide; and (b) recovering the polypeptide from the host cellculture.

[0018] The invention also provides a pharmaceutical compositioncomprising a substantially purified polypeptide having the sequence ofSEQ ID NO: 1 or a fragment of SEQ ID NO: 1 in conjunction with asuitable pharmaceutical carrier.

[0019] The invention further includes a purified antibody which binds toa polypeptide comprising the sequence of SEQ ID NO: 1 or a fragment ofSEQ ID NO: 1, as well as a purified agonist and a purified antagonist ofthe polypeptide.

[0020] The invention also provides a method for treating or preventing adisorder associated with decreased expression or activity of CJPDZ, themethod comprising administering to a subject in need of such treatmentan effective amount of a pharmaceutical composition comprising asubstantially purified polypeptide having the amino acid sequence of SEQID NO: 1 or a fragment of SEQ ID NO: 1, in conjunction with a suitablepharmaceutical carrier.

[0021] The invention also provides a method for treating or preventing adisorder associated with increased expression or activity of CJPDZ, themethod comprising administering to a subject in need of such treatmentan effective amount of an antagonist of the polypeptide having the aminoacid sequence of SEQ ID NO: 1 or a fragment of SEQ ID NO: 1.

BRIEF DESCRIPTION OF THE FIGURES AND TABLE

[0022]FIGS. 1A, 1B, and 1C show the amino acid sequence (SEQ ID NO: 1)and nucleic acid sequence (SEQ ID NO:2) of CJPDZ. The alignment wasproduced using MACDNASIS PRO software (Hitachi Software Engineering, S.San Francisco Calif.).

[0023]FIG. 2 shows the amino acid sequence alignment between residues 25through 204 of CJPDZ (1974337; SEQ ID NO:1) and residues 117 through 295of C. elegans LIN-7 (GI 1685067; SEQ ID NO:3), produced using themultisequence alignment program of LASERGENE software (DNASTAR, MadisonWis.).

[0024] Table 1 shows the programs, their descriptions, references, andthreshold parameters used to analyze CJPDZ.

DESCRIPTION OF THE INVENTION

[0025] Before the present proteins, nucleotide sequences, and methodsare described, it is understood that this invention is not limited tothe particular machines, materials and methods described, as these mayvary. It is also to be understood that the terminology used herein isfor the purpose of describing particular embodiments only, and is notintended to limit the scope of the present invention which will belimited only by the appended claims.

[0026] It must be noted that as used herein and in the appended claims,the singular forms “a,” “an,” and “the” include plural reference unlessthe context clearly dictates otherwise. Thus, for example, a referenceto “a host cell” includes a plurality of such host cells, and areference to “an antibody” is a reference to one or more antibodies andequivalents thereof known to those skilled in the art, and so forth.

[0027] Unless defined otherwise, all technical and scientific terms usedherein have the same meanings as commonly understood by one of ordinaryskill in the art to which this invention belongs. Although any machines,materials, and methods similar or equivalent to those described hereincan be used to practice or test the present invention, the preferredmachines, materials and methods are now described. All publicationsmentioned herein are cited for the purpose of describing and disclosingthe cell lines, protocols, reagents and vectors which are reported inthe publications and which might be used in connection with theinvention. Nothing herein is to be construed as an admission that theinvention is not entitled to antedate such disclosure by virtue of priorinvention.

[0028] Definitions

[0029] “CJPDZ” refers to the amino acid sequences of substantiallypurified CJPDZ obtained from any species, particularly a mammalianspecies, including bovine, ovine, porcine, murine, equine, andpreferably the human species, from any source, whether natural,synthetic, semi-synthetic, or recombinant.

[0030] The term “agonist” refers to a molecule which, when bound toCJPDZ, increases or prolongs the duration of the effect of CJPDZ.Agonists may include proteins, nucleic acids, carbohydrates, or anyother molecules which bind to and modulate the effect of CJPDZ.

[0031] An “allelic variant” is an alternative form of the gene encodingCJPDZ. Allelic variants may result from at least one mutation in thenucleic acid sequence and may result in altered mRNAs or in polypeptideswhose structure or function may or may not be altered. Any given naturalor recombinant gene may have none, one, or many allelic forms. Commonmutational changes which give rise to allelic variants are generallyascribed to natural deletions, additions, or substitutions ofnucleotides. Each of these types of changes may occur alone, or incombination with the others, one or more times in a given sequence.

[0032] “Altered” nucleic acid sequences encoding CJPDZ include thosesequences with deletions, insertions, or substitutions of differentnucleotides, resulting in a polypeptide the same as CJPDZ or apolypeptide with at least one functional characteristic of CJPDZ.Included within this definition are polymorphisms which may or may notbe readily detectable using a particular oligonucleotide probe of thepolynucleotide encoding CJPDZ, and improper or unexpected hybridizationto allelic variants, with a locus other than the normal chromosomallocus for the polynucleotide sequence encoding CJPDZ. The encodedprotein may also be “altered,” and may contain deletions, insertions, orsubstitutions of amino acid residues which produce a silent change andresult in a functionally equivalent CJPDZ. Deliberate amino acidsubstitutions may be made on the basis of similarity in polarity,charge, solubility, hydrophobicity, hydrophilicity, and/or theamphipathic nature of the residues, as long as the biological orimmunological activity of CJPDZ is retained. For example, negativelycharged amino acids may include aspartic acid and glutamic acid,positively charged amino acids may include lysine and arginine, andamino acids with uncharged polar head groups having similarhydrophilicity values may include leucine, isoleucine, and valine;glycine and alanine; asparagine and glutamine; serine and threonine; andphenylalanine and tyrosine.

[0033] The terms “amino acid” or “amino acid sequence” refer to anoligopeptide, peptide, polypeptide, or protein sequence, or a fragmentof any of these, and to naturally occurring or synthetic molecules. Inthis context, “fragments,” “immunogenic fragments,” or “antigenicfragments” refer to fragments of CJPDZ which are preferably at least 5to about 15 amino acids in length, most preferably at least 14 aminoacids, and which retain some biological activity or immunologicalactivity of CJPDZ. Where “amino acid sequence” is recited to refer to anamino acid sequence of a naturally occurring protein molecule, “aminoacid sequence” and like terms are not meant to limit the amino acidsequence to the complete native amino acid sequence associated with therecited protein molecule.

[0034] “Amplification” relates to the production of additional copies ofa nucleic acid sequence. Amplification is generally carried out usingpolymerase chain reaction (PCR) technologies well known in the art.

[0035] The term “antagonist” refers to a molecule which, when bound toCJPDZ, decreases the amount or the duration of the effect of thebiological or immunological activity of CJPDZ. Antagonists may includeproteins, nucleic acids, carbohydrates, antibodies, or any othermolecules which decrease the effect of CJPDZ.

[0036] The term “antibody” refers to intact molecules as well as tofragments thereof, such as Fab, F(ab′)₂, and Fv fragments, which arecapable of binding the epitopic determinant. Antibodies that bind CJPDZpolypeptides can be prepared using intact polypeptides or usingfragments containing small peptides of interest as the immunizingantigen. The polypeptide or oligopeptide used to immunize an animal(e.g., a mouse, a rat, or a rabbit) can be derived from the translationof RNA, or synthesized chemically, and can be conjugated to a carrierprotein if desired. Commonly used carriers that are chemically coupledto peptides include bovine serum albumin, thyroglobulin, and keyholelimpet hemocyanin (KLH). The coupled peptide is then used to immunizethe animal.

[0037] The term “antigenic determinant” refers to that fragment of amolecule (i.e., an epitope) that makes contact with a particularantibody. When a protein or a fragment of a protein is used to immunizea host animal, numerous regions of the protein may induce the productionof antibodies which bind specifically to antigenic determinants (givenregions or three-dimensional structures on the protein). An antigenicdeterminant may compete with the intact antigen (i.e., the immunogenused to elicit the immune response) for binding to an antibody.

[0038] The term “antisense” refers to any composition containing anucleic acid sequence which is complementary to the “sense” strand of aspecific nucleic acid sequence. Antisense molecules may be produced byany method including synthesis or transcription. Once introduced into acell, the complementary nucleotides combine with natural sequencesproduced by the cell to form duplexes and to block either transcriptionor translation. The designation “negative” can refer to the antisensestrand, and the designation “positive” can refer to the sense strand.

[0039] The term “biologically active,” refers to a protein havingstructural, regulatory, or biochemical functions of a naturallyoccurring molecule. Likewise, “immunologically active” refers to thecapability of the natural, recombinant, or synthetic CJPDZ, or of anyoligopeptide thereof, to induce a specific immune response inappropriate animals or cells and to bind with specific antibodies.

[0040] The terms “complementary” or “complementarity” refer to thenatural binding of polynucleotides by base pairing. For example, thesequence “5′ A-G-T 3′” bonds to the complementary sequence “3′ T-C-A5′.” Complementarity between two single-stranded molecules may be“partial,” such that only some of the nucleic acids bind, or it may be“complete,” such that total complementarity exists between the singlestranded molecules. The degree of complementarity between nucleic acidstrands has significant effects on the efficiency and strength of thehybridization between the nucleic acid strands. This is of particularimportance in amplification reactions, which depend upon binding betweennucleic acids strands, and in the design and use of peptide nucleic acid(PNA) molecules.

[0041] A “composition comprising a given polynucleotide sequence” or a“composition comprising a given amino acid sequence” refer broadly toany composition containing the given polynucleotide or amino acidsequence. The composition may comprise a dry formulation or an aqueoussolution. Compositions comprising polynucleotide sequences encodingCJPDZ or fragments of CJPDZ may be employed as hybridization probes. Theprobes may be stored in freeze-dried form and may be associated with astabilizing agent such as a carbohydrate. In hybridizations, the probemay be deployed in an aqueous solution containing salts (e.g., NaCl),detergents (e.g., sodium dodecyl sulfate; SDS), and other components(e.g., Denhardt's solution, dry milk, salmon sperm DNA, etc.).

[0042] “Consensus sequence” refers to a nucleic acid sequence which hasbeen resequenced to resolve uncalled bases, extended using XL-PCR kit(Perkin-Elmer, Norwalk Conn.) in the 5′ and/or the 3′ direction, andresequenced, or which has been assembled from the overlapping sequencesof more than one Incyte Clone using a computer program for fragmentassembly, such as the GELVIEW Fragment Assembly system (GCG, MadisonWis.). Some sequences have been both extended and assembled to producethe consensus sequence.

[0043] The term “correlates with expression of a polynucleotide”indicates that the detection of the presence of nucleic acids, the sameor related to a nucleic acid sequence encoding CJPDZ, by northernanalysis is indicative of the presence of nucleic acids encoding CJPDZin a sample, and thereby correlates with expression of the transcriptfrom the polynucleotide encoding CJPDZ.

[0044] A “deletion” refers to a change in the amino acid or nucleotidesequence that results in the absence of one or more amino acid residuesor nucleotides.

[0045] The term “derivative” refers to the chemical modification of apolypeptide sequence, or a polynucleotide sequence. Chemicalmodifications of a polynucleotide sequence can include, for example,replacement of hydrogen by an alkyl, acyl, or amino group. A derivativepolynucleotide encodes a polypeptide which retains at least onebiological or immunological function of the natural molecule. Aderivative polypeptide is one modified by glycosylation, pegylation, orany similar process that retains at least one biological orimmunological function of the polypeptide from which it was derived.

[0046] The term “similarity” refers to a degree of complementarity.There may be partial similarity or complete similarity. The word“identity” may substitute for the word “similarity.” A partiallycomplementary sequence that at least partially inhibits an identicalsequence from hybridizing to a target nucleic acid is referred to as“substantially similar.” The inhibition of hybridization of thecompletely complementary sequence to the target sequence may be examinedusing a hybridization assay (Southern or northern blot, solutionhybridization, and the like) under conditions of reduced stringency. Asubstantially similar sequence or hybridization probe will compete forand inhibit the binding of a completely similar (identical) sequence tothe target sequence under conditions of reduced stringency. This is notto say that conditions of reduced stringency are such that non-specificbinding is permitted, as reduced stringency conditions require that thebinding of two sequences to one another be a specific (i.e., aselective) interaction. The absence of non-specific binding may betested by the use of a second target sequence which lacks even a partialdegree of complementarity (e.g., less than about 30% similarity oridentity). In the absence of non-specific binding, the substantiallysimilar sequence or probe will not hybridize to the secondnon-complementary target sequence.

[0047] The phrases “percent identity” or “% identity” refer to thepercentage of sequence similarity found in a comparison of two or moreamino acid or nucleic acid sequences. Percent identity can be determinedelectronically, e.g., by using the MEGALIGN program (DNASTAR) whichcreates alignments between two or more sequences according to methodsselected by the user, e.g., the clustal method. (See, e.g., Higgins, D.G. and P. M. Sharp (1988) Gene 73:237-244.) The clustal algorithm groupssequences into clusters by examining the distances between all pairs.The clusters are aligned pairwise and then in groups. The percentagesimilarity between two amino acid sequences, e.g., sequence A andsequence B, is calculated by dividing the length of sequence A, minusthe number of gap residues in sequence A, minus the number of gapresidues in sequence B, into the sum of the residue matches betweensequence A and sequence B, times one hundred. Gaps of low or of nosimilarity between the two amino acid sequences are not included indetermining percentage similarity. Percent identity between nucleic acidsequences can also be counted or calculated by other methods known inthe art, e.g., the Jotun Hein method. (See, e.g., Hein, J. (1990)Methods Enzymol. 183:626-645.) Identity between sequences can also bedetermined by other methods known in the art, e.g., by varyinghybridization conditions.

[0048] “Human artificial chromosomes” (HACs) are linear microchromosomeswhich may contain DNA sequences of about 6 kb to 10 Mb in size, andwhich contain all of the elements required for stable mitotic chromosomesegregation and maintenance.

[0049] The term “humanized antibody” refers to antibody molecules inwhich the amino acid sequence in the non-antigen binding regions hasbeen altered so that the antibody more closely resembles a humanantibody, and still retains its original binding ability.

[0050] “Hybridization” refers to any process by which a strand ofnucleic acid binds with a complementary strand through base pairing.

[0051] The term “hybridization complex” refers to a complex formedbetween two nucleic acid sequences by virtue of the formation ofhydrogen bonds between complementary bases. A hybridization complex maybe formed in solution (e.g., Cot or Rot analysis) or formed between onenucleic acid sequence present in solution and another nucleic acidsequence immobilized on a solid support (e.g., paper, membranes,filters, chips, pins or glass slides, or any other appropriate substrateto which cells or their nucleic acids have been fixed).

[0052] The words “insertion” or “addition” refer to changes in an aminoacid or nucleotide sequence resulting in the addition of one or moreamino acid residues or nucleotides, respectively, to the sequence foundin the naturally occurring molecule.

[0053] “Immune response” can refer to conditions associated withinflammation, trauma, immune disorders, or infectious or geneticdisease, etc. These conditions can be characterized by expression ofvarious factors, e.g., cytokines, chemokines, and other signalingmolecules, which may affect cellular and systemic defense systems.

[0054] The term “microarray” refers to an arrangement of distinctpolynucleotides on a substrate.

[0055] The terms “element” or “array element” in a microarray context,refer to hybridizable polynucleotides arranged on the surface of asubstrate.

[0056] The term “modulate” refers to a change in the activity of CJPDZ.For example, modulation may cause an increase or a decrease in proteinactivity, binding characteristics, or any other biological, functional,or immunological properties of CJPDZ.

[0057] The phrases “nucleic acid” or “nucleic acid sequence” refer to anucleotide, oligonucleotide, polynucleotide, or any fragment thereof.These phrases also refer to DNA or RNA of genomic or synthetic originwhich may be single-stranded or double-stranded and may represent thesense or the antisense strand, to peptide nucleic acid (PNA), or to anyDNA-like or RNA-like material. In this context, “fragments” refers tothose nucleic acid sequences which, when translated, would producepolypeptides retaining some functional characteristic, e.g.,antigenicity, or structural domain characteristic, e.g., ATP-bindingsite, of the full-length polypeptide.

[0058] The terms “operably associated” or “operably linked” refer tofunctionally related nucleic acid sequences. A promoter is operablyassociated or operably linked with a coding sequence if the promotercontrols the translation of the encoded polypeptide. While operablyassociated or operably linked nucleic acid sequences can be contiguousand in the same reading frame, certain genetic elements, e.g., repressorgenes, are not contiguously linked to the sequence encoding thepolypeptide but still bind to operator sequences that control expressionof the polypeptide.

[0059] The term “oligonucleotide” refers to a nucleic acid sequence ofat least about 6 nucleotides to 60 nucleotides, preferably about 15 to30 nucleotides, and most preferably about 20 to 25 nucleotides, whichcan be used in PCR amplification or in a hybridization assay ormicroarray. “Oligonucleotide” is substantially equivalent to the terms“amplimer,” “primer,” “oligomer,” and “probe,” as these terms arecommonly defined in the art.

[0060] “Peptide nucleic acid” (PNA) refers to an antisense molecule oranti-gene agent which comprises an oligonucleotide of at least about 5nucleotides in length linked to a peptide backbone of amino acidresidues ending in lysine. The terminal lysine confers solubility to thecomposition. PNAs preferentially bind complementary single stranded DNAor RNA and stop transcript elongation, and may be pegylated to extendtheir lifespan in the cell.

[0061] The term “sample” is used in its broadest sense. A samplesuspected of containing nucleic acids encoding CJPDZ, or fragmentsthereof, or CJPDZ itself, may comprise a bodily fluid; an extract from acell, chromosome, organelle, or membrane isolated from a cell; a cell;genomic DNA, RNA, or cDNA, in solution or bound to a substrate; atissue; a tissue print; etc.

[0062] The terms “specific binding” or “specifically binding” refer tothat interaction between a protein or peptide and an agonist, anantibody, or an antagonist. The interaction is dependent upon thepresence of a particular structure of the protein, e.g., the antigenicdeterminant or epitope, recognized by the binding molecule. For example,if an antibody is specific for epitope “A,” the presence of apolypeptide containing the epitope A, or the presence of free unlabeledA, in a reaction containing free labeled A and the antibody will reducethe amount of labeled A that binds to the antibody.

[0063] The term “stringent conditions” refers to conditions which permithybridization between polynucleotides and the claimed polynucleotides.Stringent conditions can be defined by salt concentration, theconcentration of organic solvent, e.g., formamide, temperature, andother conditions well known in the art. In particular, stringency can beincreased by reducing the concentration of salt, increasing theconcentration of formamide, or raising the hybridization temperature.

[0064] The term “substantially purified” refers to nucleic acid or aminoacid sequences that are removed from their natural environment and areisolated or separated, and are at least about 60% free, preferably about75% free, and most preferably about 90% free from other components withwhich they are naturally associated.

[0065] A “substitution” refers to the replacement of one or more aminoacids or nucleotides by different amino acids or nucleotides,respectively.

[0066] “Substrate” refers to any suitable rigid or semi-rigid supportincluding membranes, filters, chips, slides, wafers, fibers, magnetic ornonmagnetic beads, gels, tubing, plates, polymers, microparticles andcapillaries. The substrate can have a variety of surface forms, such aswells, trenches, pins, channels and pores, to which polynucleotides orpolypeptides are bound.

[0067] “Transformation” describes a process by which exogenous DNAenters and changes a recipient cell. Transformation may occur undernatural or artificial conditions according to various methods well knownin the art, and may rely on any known method for the insertion offoreign nucleic acid sequences into a prokaryotic or eukaryotic hostcell. The method for transformation is selected based on the type ofhost cell being transformed and may include, but is not limited to,viral infection, electroporation, heat shock, lipofection, and particlebombardment. The term “transformed” cells includes stably transformedcells in which the inserted DNA is capable of replication either as anautonomously replicating plasmid or as part of the host chromosome, aswell as transiently transformed cells which express the inserted DNA orRNA for limited periods of time.

[0068] A “variant” of CJPDZ polypeptides refers to an amino acidsequence that is altered by one or more amino acid residues. The variantmay have “conservative” changes, wherein a substituted amino acid hassimilar structural or chemical properties (e.g., replacement of leucinewith isoleucine). More rarely, a variant may have “nonconservative”changes (e.g., replacement of glycine with tryptophan). Analogous minorvariations may also include amino acid deletions or insertions, or both.Guidance in determining which amino acid residues may be substituted,inserted, or deleted without abolishing biological or immunologicalactivity may be found using computer programs well known in the art, forexample, LASERGENE software (DNASTAR).

[0069] The term “variant,” when used in the context of a polynucleotidesequence, may encompass a polynucleotide sequence related to CJPDZ. Thisdefinition may also include, for example, “allelic” (as defined above),“splice,” “species,” or “polymorphic” variants. A splice variant mayhave significant identity to a reference molecule, but will generallyhave a greater or lesser number of polynucleotides due to alternatesplicing of exons during mRNA processing. The corresponding polypeptidemay possess additional functional domains or an absence of domains.Species variants are polynucleotide sequences that vary from one speciesto another. The resulting polypeptides generally will have significantamino acid identity relative to each other. A polymorphic variant is avariation in the polynucleotide sequence of a particular gene betweenindividuals of a given species. Polymorphic variants also may encompass“single nucleotide polymorphisms” (SNPs) in which the polynucleotidesequence varies by one base. The presence of SNPs may be indicative of,for example, a certain population, a disease state, or a propensity fora disease state.

[0070] The Invention

[0071] The invention is based on the discovery of a new human celljunction PDZ protein (CJPDZ), the polynucleotides encoding CJPDZ, andthe use of these compositions for the diagnosis, treatment, orprevention of cancer, neurological disorders, and developmentaldisorders.

[0072] Nucleic acids encoding the CJPDZ of the present invention wereidentified in Incyte Clone 1974337H1 from the umbilical cord mononuclearcell cDNA library (UCMCL5T01) using a computer search for nucleotideand/or amino acid sequence alignments. A consensus sequence, SEQ IDNO:2, was derived from the following overlapping and/or extended nucleicacid sequences: Incyte Clones 1974337H1 (UCMCL5T01), 4921529H1(TESTNOT11), 3606614H1 (LUNGNOT30), 1211072H1 (BRSTNOT02), and NCBInucleotide identification numbers g2063575 and g2064159.

[0073] In one embodiment, the invention encompasses a polypeptidecomprising the amino acid sequence of SEQ ID NO: 1, as shown in FIGS.1A, 1B, and 1C. CJPDZ is 233 amino acids in length and has fivepotential protein kinase C phosphorylation sites at S52, T89, S181,T189, and T205. CJPDZ has chemical and structural similarity with C.elegans LIN-7 (GI 1685067; SEQ ID NO:3). CJPDZ and LIN-7 share 53%identity overall and 69% identity within the region from L25 to R204 ofCJPDZ and from L117 to R295 of LIN-7, as shown in FIG. 2. Within thisregion, CJPDZ contains a putative PDZ domain from R107 to T189 whichshares 82% sequence identity with the PDZ domain of LIN-7. The GDdipeptide and asparagine residue found in nearly all PDZ domains areconserved in CJPDZ at G152, D153, and N159. In addition, the putativePDZ domain of CJPDZ contains eleven glycine residues, consistent withthe glycine-rich nature of nearly all PDZ domains. A fragment of SEQ IDNO:2 from about nucleotide 432 to about nucleotide 461 is useful inhybridization or amplification technologies to identify SEQ ID NO:2 andto distinguish between SEQ ID NO:2 and a related sequence. Northernanalysis shows the expression of this sequence in mononuclear cellsderived from umbilical cord blood, in the testis, in fetal lung, and inbreast tissue.

[0074] The invention also encompasses CJPDZ variants. A preferred CJPDZvariant is one which has at least about 80%, more preferably at leastabout 90%, and most preferably at least about 95% amino acid sequenceidentity to the CJPDZ amino acid sequence, and which contains at leastone functional or structural characteristic of CJPDZ.

[0075] The invention also encompasses polynucleotides which encodeCJPDZ. In a particular embodiment, the invention encompasses apolynucleotide sequence comprising the sequence of SEQ ID NO:2, whichencodes CJPDZ.

[0076] The invention also encompasses a variant of a polynucleotidesequence encoding CJPDZ. In particular, such a variant polynucleotidesequence will have at least about 70%, more preferably at least about85%, and most preferably at least about 95% polynucleotide sequenceidentity to the polynucleotide sequence encoding CJPDZ. A particularaspect of the invention encompasses a variant of SEQ ID NO:2 which hasat least about 70%, more preferably at least about 85%, and mostpreferably at least about 95% polynucleotide sequence identity to SEQ IDNO:2. Any one of the polynucleotide variants described above can encodean amino acid sequence which contains at least one functional orstructural characteristic of CJPDZ.

[0077] It will be appreciated by those skilled in the art that as aresult of the degeneracy of the genetic code, a multitude ofpolynucleotide sequences encoding CJPDZ, some bearing minimal similarityto the polynucleotide sequences of any known and naturally occurringgene, may be produced. Thus, the invention contemplates each and everypossible variation of polynucleotide sequence that could be made byselecting combinations based on possible codon choices. Thesecombinations are made in accordance with the standard triplet geneticcode as applied to the polynucleotide sequence of naturally occurringCJPDZ, and all such variations are to be considered as beingspecifically disclosed.

[0078] Although nucleotide sequences which encode CJPDZ and its variantsare preferably capable of hybridizing to the nucleotide sequence of thenaturally occurring CJPDZ under appropriately selected conditions ofstringency, it may be advantageous to produce nucleotide sequencesencoding CJPDZ or its derivatives possessing a substantially differentcodon usage, e.g., inclusion of non-naturally occurring codons. Codonsmay be selected to increase the rate at which expression of the peptideoccurs in a particular prokaryotic or eukaryotic host in accordance withthe frequency with which particular codons are utilized by the host.Other reasons for substantially altering the nucleotide sequenceencoding CJPDZ and its derivatives without altering the encoded aminoacid sequences include the production of RNA transcripts having moredesirable properties, such as a greater half-life, than transcriptsproduced from the naturally occurring sequence.

[0079] The invention also encompasses production of DNA sequences whichencode CJPDZ and CJPDZ derivatives, or fragments thereof, entirely bysynthetic chemistry. After production, the synthetic sequence may beinserted into any of the many available expression vectors and cellsystems using reagents well known in the art. Moreover, syntheticchemistry may be used to introduce mutations into a sequence encodingCJPDZ or any fragment thereof.

[0080] Also encompassed by the invention are polynucleotide sequencesthat are capable of hybridizing to the claimed polynucleotide sequences,and, in particular, to those shown in SEQ ID NO:2, or to a fragment ofSEQ ID NO:2, under various conditions of stringency. (See, e.g., Wahl,G. M. and S. L. Berger (1987) Methods Enzymol. 152:399-407; Kimmel, A.R. (1987) Methods Enzymol. 152:507-511.) For example, stringent saltconcentration will ordinarily be less than about 750 mM NaCl and 75 mMtrisodium citrate, preferably less than about 500 mM NaCl and 50 mMtrisodium citrate, and most preferably less than about 250 mM NaCl and25 mM trisodium citrate. Low stringency hybridization can be obtained inthe absence of organic solvent, e.g., formamide, while high stringencyhybridization can be obtained in the presence of at least about 35%formamide, and most preferably at least about 50% formamide. Stringenttemperature conditions will ordinarily include temperatures of at leastabout 30° C., more preferably of at least about 37° C., and mostpreferably of at least about 42° C. Varying additional parameters, suchas hybridization time, the concentration of detergent, e.g., sodiumdodecyl sulfate (SDS), and the inclusion or exclusion of carrier DNA,are well known to those skilled in the art. Various levels of stringencyare accomplished by combining these various conditions as needed. In apreferred embodiment, hybridization will occur at 30° C. in 750 mM NaCl,75 mM trisodium citrate, and 1% SDS. In a more preferred embodiment,hybridization will occur at 37° C. in 500 mM NaCl, 50 mM trisodiumcitrate, 1% SDS, 35% formamide, and 100 μg/ml denatured salmon sperm DNA(ssDNA). In a most preferred embodiment, hybridization will occur at 42°C. in 250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 50% formamide, and200 μg/ml ssDNA. Useful variations on these conditions will be readilyapparent to those skilled in the art.

[0081] The washing steps which follow hybridization can also vary instringency. Wash stringency conditions can be defined by saltconcentration and by temperature. As above, wash stringency can beincreased by decreasing salt concentration or by increasing temperature.For example, stringent salt concentration for the wash steps willpreferably be less than about 30 mM NaCl and 3 mM trisodium citrate, andmost preferably less than about 15 mM NaCl and 1.5 mM trisodium citrate.Stringent temperature conditions for the wash steps will ordinarilyinclude temperature of at least about 25° C., more preferably of atleast about 42° C., and most preferably of at least about 68° C. In apreferred embodiment, wash steps will occur at 25° C. in 30 mM NaCl, 3mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, washsteps will occur at 42° C. in mM NaCl, 1.5 mM trisodium citrate, and0.1% SDS. In a most preferred embodiment, wash steps will occur at 68°C. in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. Additionalvariations on these conditions will be readily apparent to those skilledin the art.

[0082] Methods for DNA sequencing are well known in the art and may beused to practice any of the embodiments of the invention. The methodsmay employ such enzymes as the Klenow fragment of DNA polymerase I,SEQUENASE (US Biochemical, Cleveland Ohio), Taq polymerase(Perkin-Elmer), thermostable T7 polymerase (Amersham Pharmacia Biotech,Piscataway N.J.), or combinations of polymerases and proofreadingexonucleases such as those found in the ELONGASE amplification system(Life Technologies, Gaithersburg Md.). Preferably, sequence preparationis automated with machines such as the Robbins Hydra microdispenser(Robbins Scientific, Sunnyvale Calif.), Hamilton MICROLAB 2200(Hamilton, Reno Nev.), Peltier Thermal Cycler 200 (PTC200; MJ Research,Watertown Mass.) and the ABI CATALYST 800 (Perkin-Elmer). Sequencing isthen carried out using either ABI 373 or 377 DNA sequencing systems(Perkin-Elmer) or the MEGABACE 1000 DNA sequencing system (MolecularDynamics, Sunnyvale Calif.). The resulting sequences are analyzed usinga variety of algorithms which are well known in the art. (See, e.g.,Ausubel, F. M. (1997) Short Protocols in Molecular Biology, John Wiley &Sons, New York N.Y., unit 7.7; Meyers, R. A. (1995) Molecular Biologyand Biotechnology, Wiley VCH, New York N.Y., pp. 856-853.)

[0083] The nucleic acid sequences encoding CJPDZ may be extendedutilizing a partial nucleotide sequence and employing various PCR-basedmethods known in the art to detect upstream sequences, such as promotersand regulatory elements. For example, one method which may be employed,restriction-site PCR, uses universal and nested primers to amplifyunknown sequence from genomic DNA within a cloning vector. (See, e.g.,Sarkar, G. (1993) PCR Methods Applic. 2:318-322.) Another method,inverse PCR, uses primers that extend in divergent directions to amplifyunknown sequence from a circularized template. The template is derivedfrom restriction fragments comprising a known genomic locus andsurrounding sequences. (See, e.g., Triglia, T. et al. (1988) NucleicAcids Res. 16:8186.) A third method, capture PCR, involves PCRamplification of DNA fragments adjacent to known sequences in human andyeast artificial chromosome DNA. (See, e.g., Lagerstrom, M. et al.(1991) PCR Methods Applic. 1:111-119.) In this method, multiplerestriction enzyme digestions and ligations may be used to insert anengineered double-stranded sequence into a region of unknown sequencebefore performing PCR. Other methods which may be used to retrieveunknown sequences are known in the art. (See, e.g., Parker, J. D. et al.(1991) Nucleic Acids Res. 19:3055-306). Additionally, one may use PCR,nested primers, and PROMOTERFINDER libraries (Clontech, Palo AltoCalif.) to walk genomic DNA. This procedure avoids the need to screenlibraries and is useful in finding intron/exon junctions. For allPCR-based methods, primers may be designed using commercially availablesoftware, such as OLIGO 4.06 Primer Analysis software (NationalBiosciences, Plymouth MN) or another appropriate program, to be about 22to 30 nucleotides in length, to have a GC content of about 50% or more,and to anneal to the template at temperatures of about 68° C. to 72° C.

[0084] When screening for full-length cDNAs, it is preferable to uselibraries that have been size-selected to include larger cDNAs. Inaddition, random-primed libraries, which often include sequencescontaining the 5′ regions of genes, are preferable for situations inwhich an oligo d(T) library does not yield a full-length cDNA. Genomiclibraries may be useful for extension of sequence into 5′non-transcribed regulatory regions.

[0085] Capillary electrophoresis systems which are commerciallyavailable may be used to analyze the size or confirm the nucleotidesequence of sequencing or PCR products. In particular, capillarysequencing may employ flowable polymers for electrophoretic separation,four different nucleotide-specific, laser-stimulated fluorescent dyes,and a charge coupled device camera for detection of the emittedwavelengths. Output/light intensity may be converted to electricalsignal using appropriate software (e.g., GENOTYPER and SEQUENCENAVIGATOR, Perkin-Elmer), and the entire process from loading of samplesto computer analysis and electronic data display may be computercontrolled. Capillary electrophoresis is especially preferable forsequencing small DNA fragments which may be present in limited amountsin a particular sample.

[0086] In another embodiment of the invention, polynucleotide sequencesor fragments thereof which encode CJPDZ may be cloned in recombinant DNAmolecules that direct expression of CJPDZ, or fragments or functionalequivalents thereof, in appropriate host cells. Due to the inherentdegeneracy of the genetic code, other DNA sequences which encodesubstantially the same or a functionally equivalent amino acid sequencemay be produced and used to express CJPDZ.

[0087] The nucleotide sequences of the present invention can beengineered using methods generally known in the art in order to alterCJPDZ-encoding sequences for a variety of purposes including, but notlimited to, modification of the cloning, processing, and/or expressionof the gene product. DNA shuffling by random fragmentation and PCRreassembly of gene fragments and synthetic oligonucleotides may be usedto engineer the nucleotide sequences. For example,oligonucleotide-mediated site-directed mutagenesis may be used tointroduce mutations that create new restriction sites, alterglycosylation patterns, change codon preference, produce splicevariants, and so forth.

[0088] In another embodiment, sequences encoding CJPDZ may besynthesized, in whole or in part, using chemical methods well known inthe art. (See, e.g., Caruthers, M. H. et al. (1980) Nucl. Acids Symp.Ser. 7:215-223, and Horn, T. et al. (1980) Nucl. Acids. Symp. Ser.7:225-232.) Alternatively, CJPDZ itself or a fragment thereof may besynthesized using chemical methods. For example, peptide synthesis canbe performed using various solid-phase techniques. (See, e.g., Roberge,J. Y. et al. (1995) Science 269:202-204.) Automated synthesis may beachieved using the ABI 431A Peptide Synthesizer (Perkin-Elmer).Additionally, the amino acid sequence of CJPDZ, or any part thereof, maybe altered during direct synthesis and/or combined with sequences fromother proteins, or any part thereof, to produce a variant polypeptide.

[0089] The peptide may be substantially purified by preparative highperformance liquid chromatography. (See, e.g, Chiez, R. M. and F. Z.Regnier (1990) Methods Enzymol. 182:392-421.) The composition of thesynthetic peptides may be confirmed by amino acid analysis or bysequencing. (See, e.g., Creighton, T. (1984) Proteins, Structures andMolecular Properties, WH Freeman, New York N.Y.)

[0090] In order to express a biologically active CJPDZ, the nucleotidesequences encoding CJPDZ or derivatives thereof may be inserted into anappropriate expression vector, i.e., a vector which contains thenecessary elements for transcriptional and translational control of theinserted coding sequence in a suitable host. These elements includeregulatory sequences, such as enhancers, constitutive and induciblepromoters, and 5′ and 3′ untranslated regions in the vector and inpolynucleotide sequences encoding CJPDZ. Such elements may vary in theirstrength and specificity. Specific initiation signals may also be usedto achieve more efficient translation of sequences encoding CJPDZ. Suchsignals include the ATG initiation codon and adjacent sequences, e.g.the Kozak sequence. In cases where sequences encoding CJPDZ and itsinitiation codon and upstream regulatory sequences are inserted into theappropriate expression vector, no additional transcriptional ortranslational control signals may be needed. However, in cases whereonly coding sequence, or a fragment thereof, is inserted, exogenoustranslational control signals including an in-frame ATG initiation codonshould be provided by the vector. Exogenous translational elements andinitiation codons may be of various origins, both natural and synthetic.The efficiency of expression may be enhanced by the inclusion ofenhancers appropriate for the particular host cell system used. (See,e.g., Scharf, D. et al. (1994) Results Probl. Cell Differ. 20:125-162.)

[0091] Methods which are well known to those skilled in the art may beused to construct expression vectors containing sequences encoding CJPDZand appropriate transcriptional and translational control elements.These methods include in vitro recombinant DNA techniques, synthetictechniques, and in vivo genetic recombination. (See, e.g., Sambrook, J.et al. (1989) Molecular Cloning, A Laboratory Manual, Cold Spring HarborPress, Plainview N.Y., ch. 4, 8, and 16-17; Ausubel, F. M. et al. (1995)Current Protocols in Molecular Biology, John Wiley & Sons, New YorkN.Y., ch. 9, 13, and 16.)

[0092] A variety of expression vector/host systems may be utilized tocontain and express sequences encoding CJPDZ. These include, but are notlimited to, microorganisms such as bacteria transformed with recombinantbacteriophage, plasmid, or cosmid DNA expression vectors; yeasttransformed with yeast expression vectors; insect cell systems infectedwith viral expression vectors (e.g., baculovirus); plant cell systemstransformed with viral expression vectors (e.g., cauliflower mosaicvirus, CaMV, or tobacco mosaic virus, TMV) or with bacterial expressionvectors (e.g., Ti or pBR322 plasmids); or animal cell systems. Theinvention is not limited by the host cell employed.

[0093] In bacterial systems, a number of cloning and expression vectorsmay be selected depending upon the use intended for polynucleotidesequences encoding CJPDZ. For example, routine cloning, subcloning, andpropagation of polynucleotide sequences encoding CJPDZ can be achievedusing a multifunctional E. coli vector such as PBLUESCRIPT (Stratagene,La Jolla Calif.) or pSPORT1 plasmid (Life Technologies). Ligation ofsequences encoding CJPDZ into the vector's multiple cloning sitedisrupts the lacZ gene, allowing a calorimetric screening procedure foridentification of transformed bacteria containing recombinant molecules.In addition, these vectors may be useful for in vitro transcription,dideoxy sequencing, single strand rescue with helper phage, and creationof nested deletions in the cloned sequence. (See, e.g., Van Heeke, G.and S. M. Schuster (1989) J. Biol. Chem. 264:5503-5509.) When largequantities of CJPDZ are needed, e.g. for the production of antibodies,vectors which direct high level expression of CJPDZ may be used. Forexample, vectors containing the strong, inducible T5 or T7 bacteriophagepromoter may be used.

[0094] Yeast expression systems may be used for production of CJPDZ. Anumber of vectors containing constitutive or inducible promoters, suchas alpha factor, alcohol oxidase, and PGH promoters, may be used in theyeast Saccharomyces cerevisiae or Pichia pastoris. In addition, suchvectors direct either the secretion or intracellular retention ofexpressed proteins and enable integration of foreign sequences into thehost genome for stable propagation. (See, e.g., Ausubel, 1995, supra;Grant et al. (1987) Methods Enzymol. 153:516-54; and Scorer, C. A. etal. (1994) Bio/Technology 12:181-184.)

[0095] Plant systems may also be used for expression of CJPDZ.Transcription of sequences encoding CJPDZ may be driven by viralpromoters, e.g., the 35S and 19S promoters of CaMV used alone or incombination with the omega leader sequence from TMV (Takamatsu, N.(1987) EMBO J. 6:307-311). Alternatively, plant promoters such as thesmall subunit of RUBISCO or heat shock promoters may be used. (See,e.g., Coruzzi, G. et al. (1984) EMBO J. 3:1671-1680; Broglie, R. et al.(1984) Science 224:838-843; and Winter, J. et al. (1991) Results Probl.Cell Differ. 17:85-105.) These constructs can be introduced into plantcells by direct DNA transformation or pathogen-mediated transfection.(See, e.g., The McGraw Hill Yearbook of Science and Technology (1992)McGraw Hill, New York N.Y., pp. 191-196.)

[0096] In mammalian cells, a number of viral-based expression systemsmay be utilized. In cases where an adenovirus is used as an expressionvector, sequences encoding CJPDZ may be ligated into an adenovirustranscription/translation complex consisting of the late promoter andtripartite leader sequence. Insertion in a non-essential E1 or E3 regionof the viral genome may be used to obtain infective virus whichexpresses CJPDZ in host cells. (See, e.g., Logan, J. and T. Shenk (1984)Proc. Natl. Acad. Sci. 81:3655-3659.) In addition, transcriptionenhancers, such as the Rous sarcoma virus (RSV) enhancer, may be used toincrease expression in mammalian host cells. SV40 or EBV-based vectorsmay also be used for high-level protein expression.

[0097] Human artificial chromosomes (HACs) may also be employed todeliver larger fragments of DNA than can be contained in and expressedfrom a plasmid. HACs of about 6 kb to 10 Mb are constructed anddelivered via conventional delivery methods (liposomes, polycationicamino polymers, or vesicles) for therapeutic purposes. (See, e.g.,Harrington, J. J. et al. (1997) Nat Genet. 15:345-355.)

[0098] For long term production of recombinant proteins in mammaliansystems, stable expression of CJPDZ in cell lines is preferred. Forexample, sequences encoding CJPDZ can be transformed into cell linesusing expression vectors which may contain viral origins of replicationand/or endogenous expression elements and a selectable marker gene onthe same or on a separate vector. Following the introduction of thevector, cells may be allowed to grow for about 1 to 2 days in enrichedmedia before being switched to selective media. The purpose of theselectable marker is to confer resistance to a selective agent, and itspresence allows growth and recovery of cells which successfully expressthe introduced sequences. Resistant clones of stably transformed cellsmay be propagated using tissue culture techniques appropriate to thecell type.

[0099] Any number of selection systems may be used to recovertransformed cell lines. These include, but are not limited to, theherpes simplex virus thymidine kinase and adeninephosphoribosyltransferase genes, for use in tk⁻ or apr⁻ cells,respectively. (See, e.g., Wigler, M. et al. (1977) Cell 11:223-232;Lowy, I. et al. (1980) Cell 22:817-823.) Also, antimetabolite,antibiotic, or herbicide resistance can be used as the basis forselection. For example, dhfr confers resistance to methotrexate; neoconfers resistance to the aminoglycosides, neomycin and G-418; and alsor pat confer resistance to chlorsulfuron and phosphinotricinacetyltransferase, respectively. (See, e.g., Wigler, M. et al. (1980)Proc. Natl. Acad. Sci. 77:3567-3570; Colbere-Garapin, F. et al. (1981)J. Mol. Biol. 150:1-14.) Additional selectable genes have beendescribed, e.g., trpB and hisD, which alter cellular requirements formetabolites. (See, e.g., Hartman, S. C. and R. C. Mulligan (1988) Proc.Natl. Acad. Sci. 85:8047-8051.) Visible markers, e.g., anthocyanins,green fluorescent proteins (GFP; Clontech), β glucuronidase and itssubstrate β-glucuronide, or luciferase and its substrate luciferin maybe used. These markers can be used not only to identify transformants,but also to quantify the amount of transient or stable proteinexpression attributable to a specific vector system. (See, e.g., Rhodes,C. A. (1995) Methods Mol. Biol. 55:121-131.)

[0100] Although the presence/absence of marker gene expression suggeststhat the gene of interest is also present, the presence and expressionof the gene may need to be confirmed. For example, if the sequenceencoding CJPDZ is inserted within a marker gene sequence, transformedcells containing sequences encoding CJPDZ can be identified by theabsence of marker gene function. Alternatively, a marker gene can beplaced in tandem with a sequence encoding CJPDZ under the control of asingle promoter. Expression of the marker gene in response to inductionor selection usually indicates expression of the tandem gene as well.

[0101] In general, host cells that contain the nucleic acid sequenceencoding CJPDZ and that express CJPDZ may be identified by a variety ofprocedures known to those of skill in the art. These procedures include,but are not limited to, DNA-DNA or DNA-RNA hybridizations, PCRamplification, and protein bioassay or immunoassay techniques whichinclude membrane, solution, or chip based technologies for the detectionand/or quantification of nucleic acid or protein sequences.

[0102] Immunological methods for detecting and measuring the expressionof CJPDZ using either specific polyclonal or monoclonal antibodies areknown in the art. Examples of such techniques include enzyme-linkedimmunosorbent assays (ELISAs), radioimmunoassays (RIAs), andfluorescence activated cell sorting (FACS). A two-site, monoclonal-basedimmunoassay utilizing monoclonal antibodies reactive to twonon-interfering epitopes on CJPDZ is preferred, but a competitivebinding assay may be employed. These and other assays are well known inthe art. (See, e.g., Hampton, R. et al. (1990) Serological Methods, aLaboratory Manual, APS Press, St Paul Minn., Sect. IV; Coligan, J. E. etal. (1997) Current Protocols in Immunology, Greene Pub. Associates andWiley-Interscience, New York N.Y.; and Pound, J. D. (1998)Immunochemical Protocols, Humana Press, Totowa N.J.).

[0103] A wide variety of labels and conjugation techniques are known bythose skilled in the art and may be used in various nucleic acid andamino acid assays. Means for producing labeled hybridization or PCRprobes for detecting sequences related to polynucleotides encoding CJPDZinclude oligolabeling, nick translation, end-labeling, or PCRamplification using a labeled nucleotide. Alternatively, the sequencesencoding CJPDZ, or any fragments thereof, may be cloned into a vectorfor the production of an mRNA probe. Such vectors are known in the art,are commercially available, and may be used to synthesize RNA probes invitro by addition of an appropriate RNA polymerase such as T7, T3, orSP6 and labeled nucleotides. These procedures may be conducted using avariety of commercially available kits, such as those provided byAmersham Pharmacia Biotech, Promega (Madison Wis.), and US Biochemical.Suitable reporter molecules or labels which may be used for ease ofdetection include radionuclides, enzymes, fluorescent, chemiluminescent,or chromogenic agents, as well as substrates, cofactors, inhibitors,magnetic particles, and the like.

[0104] Host cells transformed with nucleotide sequences encoding CJPDZmay be cultured under conditions suitable for the expression andrecovery of the protein from cell culture. The protein produced by atransformed cell may be secreted or retained intracellularly dependingon the sequence and/or the vector used. As will be understood by thoseof skill in the art, expression vectors containing polynucleotides whichencode CJPDZ may be designed to contain signal sequences which directsecretion of CJPDZ through a prokaryotic or eukaryotic cell membrane.

[0105] In addition, a host cell strain may be chosen for its ability tomodulate expression of the inserted sequences or to process theexpressed protein in the desired fashion. Such modifications of thepolypeptide include, but are not limited to, acetylation, carboxylation,glycosylation, phosphorylation, lipidation, and acylation.Post-translational processing which cleaves a “prepro” form of theprotein may also be used to specify protein targeting, folding, and/oractivity. Different host cells which have specific cellular machineryand characteristic mechanisms for post-translational activities (e.g.,CHO, HeLa, MDCK, HEK293, and W138), are available from the American TypeCulture Collection (ATCC, Bethesda Md.) and may be chosen to ensure thecorrect modification and processing of the foreign protein.

[0106] In another embodiment of the invention, natural, modified, orrecombinant nucleic acid sequences encoding CJPDZ may be ligated to aheterologous sequence resulting in translation of a fusion protein inany of the aforementioned host systems. For example, a chimeric CJPDZprotein containing a heterologous moiety that can be recognized by acommercially available antibody may facilitate the screening of peptidelibraries for inhibitors of CJPDZ activity. Heterologous protein andpeptide moieties may also facilitate purification of fusion proteinsusing commercially available affinity matrices. Such moieties include,but are not limited to, glutathione S-transferase (GST), maltose bindingprotein (MBP), thioredoxin (Trx), calmodulin binding peptide (CBP),6-His, FLAG, c-myc, and hemagglutinin (HA). GST, MBP, Trx, CBP, and6-His enable purification of their cognate fusion proteins onimmobilized glutathione, maltose, phenylarsine oxide, calmodulin, andmetal-chelate resins, respectively. FLAG, c-myc, and hemagglutinin (HA)enable immunoaffinity purification of fusion proteins using commerciallyavailable monoclonal and polyclonal antibodies that specificallyrecognize these epitope tags. A fusion protein may also be engineered tocontain a proteolytic cleavage site located between the CJPDZ encodingsequence and the heterologous protein sequence, so that CJPDZ may becleaved away from the heterologous moiety following purification.Methods for fusion protein expression and purification are discussed inAusubel (1995, supra, ch 10). A variety of commercially available kitsmay also be used to facilitate expression and purification of fusionproteins.

[0107] In a further embodiment of the invention, synthesis ofradiolabeled CJPDZ may be achieved in vitro using the TNT rabbitreticulocyte lysate or wheat germ extract systems (Promega). Thesesystems couple transcription and translation of protein-coding sequencesoperably associated with the T7, T3, or SP6 promoters. Translation takesplace in the presence of a radiolabeled amino acid precursor, preferably³⁵S-methionine.

[0108] Fragments of CJPDZ may be produced not only by recombinantproduction, but also by direct peptide synthesis using solid-phasetechniques. (See, e.g., Creighton, supra pp. 55-60.) Protein synthesismay be performed by manual techniques or by automation. Automatedsynthesis may be achieved, for example, using the ABI 431A PeptideSynthesizer (Perkin-Elmer). Various fragments of CJPDZ may besynthesized separately and then combined to produce the full lengthmolecule.

[0109] Therapeutics

[0110] Chemical and structural similarity, e.g., in the context ofsequences and motifs, exists between regions of CJPDZ and the LIN-7 PDZdomain-containing protein. Therefore, CJPDZ appears to be associatedwith disorders related to PDZ function, such as cancer, neurologicaldisorders, and developmental disorders. In the treatment of suchdisorders associated with increased CJPDZ expression or activity, it isdesirable to decrease the expression or activity of CJPDZ. In thetreatment of such disorders associated with decreased CJPDZ expressionor activity, it is desirable to increase the expression or activity ofCJPDZ.

[0111] Therefore, in one embodiment, CJPDZ or a fragment or derivativethereof may be administered to a subject to treat or prevent a disorderassociated with decreased expression or activity of CJPDZ.

[0112] Examples of such disorders include cancers such asadenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma,teratocarcinoma, and, in particular, cancers of the adrenal gland,bladder, bone, bone marrow, brain, breast, cervix, gall bladder,ganglia, gastrointestinal tract, heart, kidney, liver, lung, muscle,ovary, pancreas, parathyroid, penis, prostate, salivary glands, skin,spleen, testis, thymus, thyroid, and uterus; neurological disorders suchas epilepsy, ischemic cerebrovascular disease, stroke, cerebralneoplasms, Alzheimer's disease, Pick's disease, Huntington's disease,dementia, Parkinson's disease and other extrapyramidal disorders,amyotrophic lateral sclerosis and other motor neuron disorders,progressive neural muscular atrophy, retinitis pigmentosa, hereditaryataxias, multiple sclerosis and other demyelinating diseases, bacterialand viral meningitis, brain abscess, subdural empyema, epidural abscess,suppurative intracranial thrombophlebitis, myelitis and radiculitis,viral central nervous system disease, prion diseases including kuru,Creutzfeldt-Jakob disease, and Gerstmann-Straussler-Scheinker syndrome,fatal familial insomnia, nutritional and metabolic diseases of thenervous system, neurofibromatosis, tuberous sclerosis, cerebelloretinalhemangioblastomatosis, encephalotrigeminal syndrome, mental retardationand other developmental disorders of the central nervous system,cerebral palsy, neuroskeletal disorders, autonomic nervous systemdisorders, cranial nerve disorders, spinal cord diseases, musculardystrophy and other neuromuscular disorders, peripheral nervous systemdisorders, dermatomyositis and polymyositis, inherited, metabolic,endocrine, and toxic myopathies, myasthenia gravis, periodic paralysis,mental disorders including mood, anxiety, and schizophrenic disorders,akathesia, amnesia, catatonia, diabetic neuropathy, tardive dyskinesia,dystonias, paranoid psychoses, postherpetic neuralgia, and Tourette'sdisorder; and developmental disorders such as renal tubular acidosis,anemia, Cushing's syndrome, achondroplastic dwarfism, Duchenne andBecker muscular dystrophy, epilepsy, gonadal dysgenesis, WAGR syndrome(Wilms' tumor, aniridia, genitourinary abnormalities, and mentalretardation), Smith-Magenis syndrome, myelodysplastic syndrome,hereditary mucoepithelial dysplasia, hereditary keratodermas, hereditaryneuropathies such as Charcot-Marie-Tooth disease and neurofibromatosis,hypothyroidism, hydrocephalus, seizure disorders such as Syndenham'schorea and cerebral palsy, spina bifida, Williams syndrome, anencephaly,craniorachischisis, congenital glaucoma, cataract, sensorineural hearingloss, and any disorder associated with cell growth and differentiation,embryogenesis, and morphogenesis involving any tissue, organ, or systemof a subject, e.g., the brain, adrenal gland, kidney, skeletal orreproductive system.

[0113] In another embodiment, a vector capable of expressing CJPDZ or afragment or derivative thereof may be administered to a subject to treator prevent a disorder associated with decreased expression or activityof CJPDZ including, but not limited to, those described above.

[0114] In a further embodiment, a pharmaceutical composition comprisinga substantially purified CJPDZ in conjunction with a suitablepharmaceutical carrier may be administered to a subject to treat orprevent a disorder associated with decreased expression or activity ofCJPDZ including, but not limited to, those provided above.

[0115] In still another embodiment, an agonist which modulates theactivity of CJPDZ may be administered to a subject to treat or prevent adisorder associated with decreased expression or activity of CJPDZincluding, but not limited to, those listed above.

[0116] In a further embodiment, an antagonist of CJPDZ may beadministered to a subject to treat or prevent a disorder associated withincreased expression or activity of CJPDZ. Such disorders may include,but are not limited to, the cancers, neurological disorders, anddevelopmental disorders discussed above. In one aspect, an antibodywhich specifically binds CJPDZ may be used directly as an antagonist orindirectly as a targeting or delivery mechanism for bringing apharmaceutical agent to cells or tissue which express CJPDZ.

[0117] In an additional embodiment, a vector expressing the complementof the polynucleotide encoding CJPDZ may be administered to a subject totreat or prevent a disorder associated with increased expression oractivity of CJPDZ including, but not limited to, those described above.

[0118] In other embodiments, any of the proteins, antagonists,antibodies, agonists, complementary sequences, or vectors of theinvention may be administered in combination with other appropriatetherapeutic agents. Selection of the appropriate agents for use incombination therapy may be made by one of ordinary skill in the art,according to conventional pharmaceutical principles. The combination oftherapeutic agents may act synergistically to effect the treatment orprevention of the various disorders described above. Using thisapproach, one may be able to achieve therapeutic efficacy with lowerdosages of each agent, thus reducing the potential for adverse sideeffects.

[0119] An antagonist of CJPDZ may be produced using methods which aregenerally known in the art. In particular, purified CJPDZ may be used toproduce antibodies or to screen libraries of pharmaceutical agents toidentify those which specifically bind CJPDZ. Antibodies to CJPDZ mayalso be generated using methods that are well known in the art. Suchantibodies may include, but are not limited to, polyclonal, monoclonal,chimeric, and single chain antibodies, Fab fragments, and fragmentsproduced by a Fab expression library. Neutralizing antibodies (i.e.,those which inhibit dimer formation) are especially preferred fortherapeutic use.

[0120] For the production of antibodies, various hosts including goats,rabbits, rats, mice, humans, and others may be immunized by injectionwith CJPDZ or with any fragment or oligopeptide thereof which hasimmunogenic properties. Depending on the host species, various adjuvantsmay be used to increase immunological response. Such adjuvants include,but are not limited to, Freund's, mineral gels such as aluminumhydroxide, and surface active substances such as lysolecithin, pluronicpolyols, polyanions, peptides, oil emulsions, KLH, and dinitrophenol.Among adjuvants used in humans, BCG (bacilli Calmette-Guerin) andCorynebacterium parvum are especially preferable.

[0121] It is preferred that the oligopeptides, peptides, or fragmentsused to induce antibodies to CJPDZ have an amino acid sequenceconsisting of at least about 5 amino acids, and, more preferably, of atleast about 10 amino acids. It is also preferable that theseoligopeptides, peptides, or fragments are identical to a portion of theamino acid sequence of the natural protein and contain the entire aminoacid sequence of a small, naturally occurring molecule. Short stretchesof CJPDZ amino acids may be fused with those of another protein, such asKLH, and antibodies to the chimeric molecule may be produced.

[0122] Monoclonal antibodies to CJPDZ may be prepared using anytechnique which provides for the production of antibody molecules bycontinuous cell lines in culture. These include, but are not limited to,the hybridoma technique, the human B-cell hybridoma technique, and theEBV-hybridoma technique. (See, e.g., Kohler, G. et al. (1975) Nature256:495-497; Kozbor, D. et al. (1985) J. Immunol. Methods 81:31-42;Cote, R. J. et al. (1983) Proc. Natl. Acad. Sci. 80:2026-2030; and Cole,S. P. et al. (1984) Mol. Cell Biol. 62:109-120.)

[0123] In addition, techniques developed for the production of “chimericantibodies,” such as the splicing of mouse antibody genes to humanantibody genes to obtain a molecule with appropriate antigen specificityand biological activity, can be used. (See, e.g., Morrison, S. L. et al.(1984) Proc. Natl. Acad. Sci. 81:6851-6855; Neuberger, M. S. et al.(1984) Nature 312:604-608; and Takeda, S. et al. (1985) Nature314:452454.) Alternatively, techniques described for the production ofsingle chain antibodies may be adapted, using methods known in the art,to produce CJPDZ-specific single chain antibodies. Antibodies withrelated specificity, but of distinct idiotypic composition, may begenerated by chain shuffling from random combinatorial immunoglobulinlibraries. (See, e.g., Burton D. R. (1991) Proc. Natl. Acad. Sci.88:10134-10137.)

[0124] Antibodies may also be produced by inducing in vivo production inthe lymphocyte population or by screening immunoglobulin libraries orpanels of highly specific binding reagents as disclosed in theliterature. (See, e.g., Orlandi, R. et al. (1989) Proc. Natl. Acad. Sci.86: 3833-3837; Winter, G. et al. (1991) Nature 349:293-299.)

[0125] Antibody fragments which contain specific binding sites for CJPDZmay also be generated. For example, such fragments include, but are notlimited to, F(ab′)2 fragments produced by pepsin digestion of theantibody molecule and Fab fragments generated by reducing the disulfidebridges of the F(ab′)2 fragments. Alternatively, Fab expressionlibraries may be constructed to allow rapid and easy identification ofmonoclonal Fab fragments with the desired specificity. (See, e.g., Huse,W. D. et al. (1989) Science 246:1275-1281.)

[0126] Various immunoassays may be used for screening to identifyantibodies having the desired specificity. Numerous protocols forcompetitive binding or immunoradiometric assays using either polyclonalor monoclonal antibodies with established specificities are well knownin the art. Such immunoassays typically involve the measurement ofcomplex formation between CJPDZ and its specific antibody. A two-site,monoclonal-based immunoassay utilizing monoclonal antibodies reactive totwo non-interfering CJPDZ epitopes is preferred, but a competitivebinding assay may also be employed (Pound, supra).

[0127] Various methods such as Scatchard analysis in conjunction withradioimmunoassay techniques may be used to assess the affinity ofantibodies for CJPDZ. Affinity is expressed as an association constant,K_(a), which is defined as the molar concentration of CJPDZ-antibodycomplex divided by the molar concentrations of free antigen and freeantibody under equilibrium conditions. The K_(a) determined for apreparation of polyclonal antibodies, which are heterogeneous in theiraffinities for multiple CJPDZ epitopes, represents the average affinity,or avidity, of the antibodies for CJPDZ. The K_(a) determined for apreparation of monoclonal antibodies, which are monospecific for aparticular CJPDZ epitope, represents a true measure of affinity.High-affinity antibody preparations with K_(a) ranging from about 10⁹ to10¹² l/mole are preferred for use in immunoassays in which theCJPDZ-antibody complex must withstand rigorous manipulations.Low-affinity antibody preparations with K_(a) ranging from about 10⁶ to10⁷ l/mole are preferred for use in immunopurification and similarprocedures which ultimately require dissociation of CJPDZ, preferably inactive form, from the antibody (Catty, D. (1988) Antibodies, Volume I: APractical Approach, IRL Press, Washington D.C.; Liddell, J. E. andCryer, A. (1991) A Practical Guide to Monoclonal Antibodies, John Wiley& Sons, New York N.Y.).

[0128] The titer and avidity of polyclonal antibody preparations may befurther evaluated to determine the quality and suitability of suchpreparations for certain downstream applications. For example, apolyclonal antibody preparation containing at least 1-2 mg specificantibody/ml, preferably 5-10 mg specific antibody/ml, is preferred foruse in procedures requiring precipitation of CJPDZ-antibody complexes.Procedures for evaluating antibody specificity, titer, and avidity, andguidelines for antibody quality and usage in various applications, aregenerally available. (See, e.g., Catty, supra, and Coligan et al.supra.)

[0129] In another embodiment of the invention, the polynucleotidesencoding CJPDZ, or any fragment or complement thereof, may be used fortherapeutic purposes. In one aspect, the complement of thepolynucleotide encoding CJPDZ may be used in situations in which itwould be desirable to block the transcription of the mRNA. Inparticular, cells may be transformed with sequences complementary topolynucleotides encoding CJPDZ. Thus, complementary molecules orfragments may be used to modulate CJPDZ activity, or to achieveregulation of gene function. Such technology is now well known in theart, and sense or antisense oligonucleotides or larger fragments can bedesigned from various locations along the coding or control regions ofsequences encoding CJPDZ.

[0130] Expression vectors derived from retroviruses, adenoviruses, orherpes or vaccinia viruses, or from various bacterial plasmids, may beused for delivery of nucleotide sequences to the targeted organ, tissue,or cell population. Methods which are well known to those skilled in theart can be used to construct vectors to express nucleic acid sequencescomplementary to the polynucleotides encoding CJPDZ. (See, e.g.,Sambrook, supra; Ausubel, 1995, supra.)

[0131] Genes encoding CJPDZ can be turned off by transforming a cell ortissue with expression vectors which express high levels of apolynucleotide, or fragment thereof, encoding CJPDZ. Such constructs maybe used to introduce untranslatable sense or antisense sequences into acell. Even in the absence of integration into the DNA, such vectors maycontinue to transcribe RNA molecules until they are disabled byendogenous nucleases. Transient expression may last for a month or morewith a non-replicating vector, and may last even longer if appropriatereplication elements are part of the vector system.

[0132] As mentioned above, modifications of gene expression can beobtained by designing complementary sequences or antisense molecules(DNA, RNA, or PNA) to the control, 5′, or regulatory regions of the geneencoding CJPDZ. Oligonucleotides derived from the transcriptioninitiation site, e.g., between about positions −10 and +10 from thestart site, are preferred. Similarly, inhibition can be achieved usingtriple helix base-pairing methodology. Triple helix pairing is usefulbecause it causes inhibition of the ability of the double helix to opensufficiently for the binding of polymerases, transcription factors, orregulatory molecules. Recent therapeutic advances using triplex DNA havebeen described in the literature. (See, e.g., Gee, J. E. et al. (1994)in Huber, B. E. and B. I. Carr, Molecular and Immunologic Approaches,Futura Publishing, Mt. Kisco N.Y., pp. 163-177.) A complementarysequence or antisense molecule may also be designed to block translationof mRNA by preventing the transcript from binding to ribosomes.

[0133] Ribozymes, enzymatic RNA molecules, may also be used to catalyzethe specific cleavage of RNA. The mechanism of ribozyme action involvessequence-specific hybridization of the ribozyme molecule tocomplementary target RNA, followed by endonucleolytic cleavage. Forexample, engineered hammerhead motif ribozyme molecules may specificallyand efficiently catalyze endonucleolytic cleavage of sequences encodingCJPDZ.

[0134] Specific ribozyme cleavage sites within any potential RNA targetare initially identified by scanning the target molecule for ribozymecleavage sites, including the following sequences: GUA, GUU, and GUC.Once identified, short RNA sequences of between 15 and 20ribonucleotides, corresponding to the region of the target genecontaining the cleavage site, may be evaluated for secondary structuralfeatures which may render the oligonucleotide inoperable. Thesuitability of candidate targets may also be evaluated by testingaccessibility to hybridization with complementary oligonucleotides usingribonuclease protection assays.

[0135] Complementary ribonucleic acid molecules and ribozymes of theinvention may be prepared by any method known in the art for thesynthesis of nucleic acid molecules. These include techniques forchemically synthesizing oligonucleotides such as solid phasephosphoramidite chemical synthesis. Alternatively, RNA molecules may begenerated by in vitro and in vivo transcription of DNA sequencesencoding CJPDZ. Such DNA sequences may be incorporated into a widevariety of vectors with suitable RNA polymerase promoters such as T7 orSP6. Alternatively, these cDNA constructs that synthesize complementaryRNA, constitutively or inducibly, can be introduced into cell lines,cells, or tissues.

[0136] RNA molecules may be modified to increase intracellular stabilityand half-life. Possible modifications include, but are not limited to,the addition of flanking sequences at the 5′ and/or 3′ ends of themolecule, or the use of phosphorothioate or 2′ O-methyl rather thanphosphodiesterase linkages within the backbone of the molecule. Thisconcept is inherent in the production of PNAs and can be extended in allof these molecules by the inclusion of nontraditional bases such asinosine, queosine, and wybutosine, as well as acetyl-, methyl-, thio-,and similarly modified forms of adenine, cytidine, guanine, thymine, anduridine which are not as easily recognized by endogenous endonucleases.

[0137] Many methods for introducing vectors into cells or tissues areavailable and equally suitable for use in vivo, in vitro, and ex vivo.For ex vivo therapy, vectors may be introduced into stem cells takenfrom the patient and clonally propagated for autologous transplant backinto that same patient. Delivery by transfection, by liposomeinjections, or by polycationic amino polymers may be achieved usingmethods which are well known in the art. (See, e.g., Goldman, C. K. etal. (1997) Nature Biotechnology 15:462-466.)

[0138] Any of the therapeutic methods described above may be applied toany subject in need of such therapy, including, for example, mammalssuch as dogs, cats, cows, horses, rabbits, monkeys, and most preferably,humans.

[0139] An additional embodiment of the invention relates to theadministration of a pharmaceutical or sterile composition, inconjunction with a pharmaceutically acceptable carrier, for any of thetherapeutic effects discussed above. Such pharmaceutical compositionsmay consist of CJPDZ, antibodies to CJPDZ, and mimetics, agonists,antagonists, or inhibitors of CJPDZ. The compositions may beadministered alone or in combination with at least one other agent, suchas a stabilizing compound, which may be administered in any sterile,biocompatible pharmaceutical carrier including, but not limited to,saline, buffered saline, dextrose, and water. The compositions may beadministered to a patient alone, or in combination with other agents,drugs, or hormones.

[0140] The pharmaceutical compositions utilized in this invention may beadministered by any number of routes including, but not limited to,oral, intravenous, intramuscular, intra-arterial, intramedullary,intrathecal, intraventricular, transdermal, subcutaneous,intraperitoneal, intranasal, enteral, topical, sublingual, or rectalmeans.

[0141] In addition to the active ingredients, these pharmaceuticalcompositions may contain suitable pharmaceutically-acceptable carrierscomprising excipients and auxiliaries which facilitate processing of theactive compounds into preparations which can be used pharmaceutically.Further details on techniques for formulation and administration may befound in the latest edition of Remington's Pharmaceutical Sciences(Maack Publishing, Easton Pa.).

[0142] Pharmaceutical compositions for oral administration can beformulated using pharmaceutically acceptable carriers well known in theart in dosages suitable for oral administration. Such carriers enablethe pharmaceutical compositions to be formulated as tablets, pills,dragees, capsules, liquids, gels, syrups, slurries, suspensions, and thelike, for ingestion by the patient. Pharmaceutical preparations for oraluse can be obtained through combining active compounds with solidexcipient and processing the resultant mixture of granules (optionally,after grinding) to obtain tablets or dragee cores. Suitable auxiliariescan be added, if desired. Suitable excipients include carbohydrate orprotein fillers, such as sugars, including lactose, sucrose, mannitol,and sorbitol; starch from corn, wheat, rice, potato, or other plants;cellulose, such as methyl cellulose, hydroxypropylmethyl-cellulose, orsodium carboxymethylcellulose; gums, including arabic and tragacanth;and proteins, such as gelatin and collagen. If desired, disintegratingor solubilizing agents may be added, such as the cross-linked polyvinylpyrrolidone, agar, and alginic acid or a salt thereof, such as sodiumalginate.

[0143] Dragee cores may be used in conjunction with suitable coatings,such as concentrated sugar solutions, which may also contain gum arabic,talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/ortitanium dioxide, lacquer solutions, and suitable organic solvents orsolvent mixtures. Dyestuffs or pigments may be added to the tablets ordragee coatings for product identification or to characterize thequantity of active compound, i.e., dosage.

[0144] Pharmaceutical preparations which can be used orally includepush-fit capsules made of gelatin, as well as soft, sealed capsules madeof gelatin and a coating, such as glycerol or sorbitol. Push-fitcapsules can contain active ingredients mixed with fillers or binders,such as lactose or starches, lubricants, such as talc or magnesiumstearate, and, optionally, stabilizers. In soft capsules, the activecompounds may be dissolved or suspended in suitable liquids, such asfatty oils, liquid, or liquid polyethylene glycol with or withoutstabilizers.

[0145] Pharmaceutical formulations suitable for parenteraladministration may be formulated in aqueous solutions, preferably inphysiologically compatible buffers such as Hanks' solution, Ringer'ssolution, or physiologically buffered saline. Aqueous injectionsuspensions may contain substances which increase the viscosity of thesuspension, such as sodium carboxymethyl cellulose, sorbitol, ordextran. Additionally, suspensions of the active compounds may beprepared as appropriate oily injection suspensions. Suitable lipophilicsolvents or vehicles include fatty oils, such as sesame oil, orsynthetic fatty acid esters, such as ethyl oleate, triglycerides, orliposomes. Non-lipid polycationic amino polymers may also be used fordelivery. Optionally, the suspension may also contain suitablestabilizers or agents to increase the solubility of the compounds andallow for the preparation of highly concentrated solutions.

[0146] For topical or nasal administration, penetrants appropriate tothe particular barrier to be permeated are used in the formulation. Suchpenetrants are generally known in the art.

[0147] The pharmaceutical compositions of the present invention may bemanufactured in a manner that is known in the art, e.g., by means ofconventional mixing, dissolving, granulating, dragee-making, levigating,emulsifying, encapsulating, entrapping, or lyophilizing processes.

[0148] The pharmaceutical composition may be provided as a salt and canbe formed with many acids, including but not limited to, hydrochloric,sulfuric, acetic, lactic, tartaric, malic, and succinic acid. Salts tendto be more soluble in aqueous or other protonic solvents than are thecorresponding free base forms. In other cases, the preferred preparationmay be a lyophilized powder which may contain any or all of thefollowing: 1 mM to 50 mM histidine, 0.1% to 2% sucrose, and 2% to 7%mannitol, at a pH range of 4.5 to 5.5, that is combined with bufferprior to use.

[0149] After pharmaceutical compositions have been prepared, they can beplaced in an appropriate container and labeled for treatment of anindicated condition. For administration of CJPDZ, such labeling wouldinclude amount, frequency, and method of administration.

[0150] Pharmaceutical compositions suitable for use in the inventioninclude compositions wherein the active ingredients are contained in aneffective amount to achieve the intended purpose. The determination ofan effective dose is well within the capability of those skilled in theart.

[0151] For any compound, the therapeutically effective dose can beestimated initially either in cell culture assays, e.g., of neoplasticcells or in animal models such as mice, rats, rabbits, dogs, or pigs. Ananimal model may also be used to determine the appropriate concentrationrange and route of administration. Such information can then be used todetermine useful doses and routes for administration in humans.

[0152] A therapeutically effective dose refers to that amount of activeingredient, for example CJPDZ or fragments thereof, antibodies of CJPDZ,and agonists, antagonists or inhibitors of CJPDZ, which ameliorates thesymptoms or condition. Therapeutic efficacy and toxicity may bedetermined by standard pharmaceutical procedures in cell cultures orwith experimental animals, such as by calculating the ED₅₀ (the dosetherapeutically effective in 50% of the population) or LD 50 (the doselethal to 50% of the population) statistics. The dose ratio of toxic totherapeutic effects is the therapeutic index, and it can be expressed asthe LD₅₀/ED₅₀ ratio. Pharmaceutical compositions which exhibit largetherapeutic indices are preferred. The data obtained from cell cultureassays and animal studies are used to formulate a range of dosage forhuman use. The dosage contained in such compositions is preferablywithin a range of circulating concentrations that includes the ED₅₀ withlittle or no toxicity. The dosage varies within this range dependingupon the dosage form employed, the sensitivity of the patient, and theroute of administration.

[0153] The exact dosage will be determined by the practitioner, in lightof factors related to the subject requiring treatment. Dosage andadministration are adjusted to provide sufficient levels of the activemoiety or to maintain the desired effect. Factors which may be takeninto account include the severity of the disease state, the generalhealth of the subject, the age, weight, and gender of the subject, timeand frequency of administration, drug combination(s), reactionsensitivities, and response to therapy. Long-acting pharmaceuticalcompositions may be administered every 3 to 4 days, every week, orbiweekly depending on the half-life and clearance rate of the particularformulation.

[0154] Normal dosage amounts may vary from about 0.1 μg to 100,000 μg,up to a total dose of about 1 1.5 gram, depending upon the route ofadministration. Guidance as to particular dosages and methods ofdelivery is provided in the literature and generally available topractitioners in the art. Those skilled in the art will employ differentformulations for nucleotides than for proteins or their inhibitors.Similarly, delivery of polynucleotides or polypeptides will be specificto particular cells, conditions, locations, etc.

[0155] Diagnostics

[0156] In another embodiment, antibodies which specifically bind CJPDZmay be used for the diagnosis of disorders characterized by expressionof CJPDZ, or in assays to monitor patients being treated with CJPDZ oragonists, antagonists, or inhibitors of CJPDZ. Antibodies useful fordiagnostic purposes may be prepared in the same manner as describedabove for therapeutics. Diagnostic assays for CJPDZ include methodswhich utilize the antibody and a label to detect CJPDZ in human bodyfluids or in extracts of cells or tissues. The antibodies may be usedwith or without modification, and may be labeled by covalent ornon-covalent attachment of a reporter molecule. A wide variety ofreporter molecules, several of which are described above, are known inthe art and may be used.

[0157] A variety of protocols for measuring CJPDZ, including ELISAs,RIAs, and FACS, are known in the art and provide a basis for diagnosingaltered or abnormal levels of CJPDZ expression. Normal or standardvalues for CJPDZ expression are established by combining body fluids orcell extracts taken from normal mammalian subjects, preferably human,with antibody to CJPDZ under conditions suitable for complex formation.The amount of standard complex formation may be quantitated by variousmethods, preferably by photometric means. Quantities of CJPDZ expressedin subject samples, control and disease, from biopsied tissues arecompared with the standard values. Deviation between standard andsubject values establishes the parameters for diagnosing disease.

[0158] In another embodiment of the invention, the polynucleotidesencoding CJPDZ may be used for diagnostic purposes. The polynucleotideswhich may be used include oligonucleotide sequences, complementary RNAand DNA molecules, and PNAs. The polynucleotides may be used to detectand quantitate gene expression in biopsied tissues in which expressionof CJPDZ may be correlated with disease. The diagnostic assay may beused to determine absence, presence, and excess expression of CJPDZ, andto monitor regulation of CJPDZ levels during therapeutic intervention.

[0159] In one aspect, hybridization with PCR probes which are capable ofdetecting polynucleotide sequences, including genomic sequences,encoding CJPDZ or closely related molecules may be used to identifynucleic acid sequences which encode CJPDZ. The specificity of the probe,whether it is made from a highly specific region, e.g., the 5′regulatory region, or from a less specific region, e.g., a conservedmotif, and the stringency of the hybridization or amplification(maximal, high, intermediate, or low), will determine whether the probeidentifies only naturally occurring sequences encoding CJPDZ, allelicvariants, or related sequences.

[0160] Probes may also be used for the detection of related sequences,and should preferably have at least 50% sequence identity to any of theCJPDZ encoding sequences. The hybridization probes of the subjectinvention may be DNA or RNA and may be derived from the sequence of SEQID NO:2 or from genomic sequences including promoters, enhancers, andintrons of the CJPDZ gene.

[0161] Means for producing specific hybridization probes for DNAsencoding CJPDZ include the cloning of polynucleotide sequences encodingCJPDZ or CJPDZ derivatives into vectors for the production of mRNAprobes. Such vectors are known in the art, are commercially available,and may be used to synthesize RNA probes in vitro by means of theaddition of the appropriate RNA polymerases and the appropriate labelednucleotides. Hybridization probes may be labeled by a variety ofreporter groups, for example, by radionuclides such as ³²p or ³⁵S, or byenzymatic labels, such as alkaline phosphatase coupled to the probe viaavidin/biotin coupling systems, and the like.

[0162] Polynucleotide sequences encoding CJPDZ may be used for thediagnosis of disorders associated with expression of CJPDZ. Examples ofsuch disorders include, but are not limited to, cancers such asadenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma,teratocarcinoma, and, in particular, cancers of the adrenal gland,bladder, bone, bone marrow, brain, breast, cervix, gall bladder,ganglia, gastrointestinal tract, heart, kidney, liver, lung, muscle,ovary, pancreas, parathyroid, penis, prostate, salivary glands, skin,spleen, testis, thymus, thyroid, and uterus; neurological disorders suchas epilepsy, ischemic cerebrovascular disease, stroke, cerebralneoplasms, Alzheimer's disease, Pick's disease, Huntington's disease,dementia, Parkinson's disease and other extrapyramidal disorders,amyotrophic lateral sclerosis and other motor neuron disorders,progressive neural muscular atrophy, retinitis pigmentosa, hereditaryataxias, multiple sclerosis and other demyelinating diseases, bacterialand viral meningitis, brain abscess, subdural empyema, epidural abscess,suppurative intracranial thrombophlebitis, myelitis and radiculitis,viral central nervous system disease, prion diseases including kuru,Creutzfeldt-Jakob disease, and Gerstmann-Straussler-Scheinker syndrome,fatal familial insomnia, nutritional and metabolic diseases of thenervous system, neurofibromatosis, tuberous sclerosis, cerebelloretinalhemangioblastomatosis, encephalotrigeminal syndrome, mental retardationand other developmental disorders of the central nervous system,cerebral palsy, neuroskeletal disorders, autonomic nervous systemdisorders, cranial nerve disorders, spinal cord diseases, musculardystrophy and other neuromuscular disorders, peripheral nervous systemdisorders, dermatomyositis and polymyositis, inherited, metabolic,endocrine, and toxic myopathies, myasthenia gravis, periodic paralysis,mental disorders including mood, anxiety, and schizophrenic disorders,akathesia, amnesia, catatonia, diabetic neuropathy, tardive dyskinesia,dystonias, paranoid psychoses, postherpetic neuralgia, and Tourette'sdisorder; and developmental disorders such as renal tubular acidosis,anemia, Cushing's syndrome, achondroplastic dwarfism, Duchenne andBecker muscular dystrophy, epilepsy, gonadal dysgenesis, WAGR syndrome(Wilms' tumor, aniridia, genitourinary abnormalities, and mentalretardation), Smith-Magenis syndrome, myelodysplastic syndrome,hereditary mucoepithelial dysplasia, hereditary keratodermas, hereditaryneuropathies such as Charcot-Marie-Tooth disease and neurofibromatosis,hypothyroidism, hydrocephalus, seizure disorders such as Syndenham'schorea and cerebral palsy, spina bifida, Williams syndrome, anencephaly,craniorachischisis, congenital glaucoma, cataract, sensorineural hearingloss, and any disorder associated with cell growth and differentiation,embryogenesis, and morphogenesis involving any tissue, organ, or systemof a subject, e.g., the brain, adrenal gland, kidney, skeletal orreproductive system. The polynucleotide sequences encoding CJPDZ may beused in Southern or northern analysis, dot blot, or other membrane-basedtechnologies; in PCR technologies; in dipstick, pin, and multiformatELISA-like assays; and in microarrays utilizing fluids or tissues frompatients to detect altered CJPDZ expression. Such qualitative orquantitative methods are well known in the art.

[0163] In a particular aspect, the nucleotide sequences encoding CJPDZmay be useful in assays that detect the presence of associateddisorders, particularly those mentioned above. The nucleotide sequencesencoding CJPDZ may be labeled by standard methods and added to a fluidor tissue sample from a patient under conditions suitable for theformation of hybridization complexes. After a suitable incubationperiod, the sample is washed and the signal is quantitated and comparedwith a standard value. If the amount of signal in the patient sample issignificantly altered in comparison to a control sample then thepresence of altered levels of nucleotide sequences encoding CJPDZ in thesample indicates the presence of the associated disorder. Such assaysmay also be used to evaluate the efficacy of a particular therapeutictreatment regimen in animal studies, in clinical trials, or to monitorthe treatment of an individual patient.

[0164] In order to provide a basis for the diagnosis of a disorderassociated with expression of CJPDZ, a normal or standard profile forexpression is established. This may be accomplished by combining bodyfluids or cell extracts taken from normal subjects, either animal orhuman, with a sequence, or a fragment thereof, encoding CJPDZ, underconditions suitable for hybridization or amplification. Standardhybridization may be quantified by comparing the values obtained fromnormal subjects with values from an experiment in which a known amountof a substantially purified polynucleotide is used. Standard valuesobtained in this manner may be compared with values obtained fromsamples from patients who are symptomatic for a disorder. Deviation fromstandard values is used to establish the presence of a disorder.

[0165] Once the presence of a disorder is established and a treatmentprotocol is initiated, hybridization assays may be repeated on a regularbasis to determine if the level of expression in the patient begins toapproximate that which is observed in the normal subject. The resultsobtained from successive assays may be used to show the efficacy oftreatment over a period ranging from several days to months. Withrespect to cancer, the presence of an abnormal amount of transcript(either under- or over-expressed) in biopsied tissue from an individualmay indicate a predisposition for the development of the disease, or mayprovide a means for detecting the disease prior to the appearance ofactual clinical symptoms. A more definitive diagnosis of this type mayallow health professionals to employ preventative measures or aggressivetreatment earlier thereby preventing the development or furtherprogression of the cancer.

[0166] Additional diagnostic uses for oligonucleotides designed from thesequences encoding CJPDZ may involve the use of PCR. These oligomers maybe chemically synthesized, generated enzymatically, or produced invitro. Oligomers will preferably contain a fragment of a polynucleotideencoding CJPDZ, or a fragment of a polynucleotide complementary to thepolynucleotide encoding CJPDZ, and will be employed under optimizedconditions for identification of a specific gene or condition. Oligomersmay also be employed under less stringent conditions for detection orquantitation of closely related DNA or RNA sequences.

[0167] Methods which may also be used to quantitate the expression ofCJPDZ include radiolabeling or biotinylating nucleotides,coamplification of a control nucleic acid, and interpolating resultsfrom standard curves. (See, e.g., Melby, P. C. et al. (1993) J. Immunol.Methods 159:235-244; Duplaa, C. et al. (1993) Anal. Biochem. 229-236.)The speed of quantitation of multiple samples may be accelerated byrunning the assay in an ELISA format where the oligomer of interest ispresented in various dilutions and a spectrophotometric or colorimetricresponse gives rapid quantitation.

[0168] In further embodiments, oligonucleotides or longer fragmentsderived from any of the polynucleotide sequences described herein may beused as targets in a microarray. The microarray can be used to monitorthe expression level of large numbers of genes simultaneously and toidentify genetic variants, mutations, and polymorphisms. Thisinformation may be used to determine gene function, to understand thegenetic basis of a disorder, to diagnose a disorder, and to develop andmonitor the activities of therapeutic agents.

[0169] Microarrays may be prepared, used, and analyzed using methodsknown in the art. (See, e.g., Brennan, T. M. et al. (1995) U.S. Pat. No.5,474,796; Schena, M. et al. (1996) Proc. Natl. Acad. Sci.93:10614-10619; Baldeschweiler et al. (1995) PCT applicationWO95/251116; Shalon, D. et al. (1995) PCT application WO95/35505;Heller, R. A. et al. (1997) Proc. Natl. Acad. Sci. 94:2150-2155; andHeller, M. J. et al. (1997) U.S. Pat. No. 5,605,662.)

[0170] In another embodiment of the invention, nucleic acid sequencesencoding CJPDZ may be used to generate hybridization probes useful inmapping the naturally occurring genomic sequence. The sequences may bemapped to a particular chromosome, to a specific region of a chromosome,or to artificial chromosome constructions, e.g., human artificialchromosomes (HACs), yeast artificial chromosomes (YACs), bacterialartificial chromosomes (BACs), bacterial P1 constructions, or singlechromosome cDNA libraries. (See, e.g., Harrington, J. J. et al. (1997)Nat Genet. 15:345-355; Price, C. M. (1993) Blood Rev. 7:127-134; andTrask, B. J. (1991) Trends Genet. 7:149-154.)

[0171] Fluorescent in situ hybridization (FISH) may be correlated withother physical chromosome mapping techniques and genetic map data. (See,e.g., Heinz-Ulrich, et al. (1995) in Meyers, supra, pp. 965-968.)Examples of genetic map data can be found in various scientific journalsor at the Online Mendelian Inheritance in Man (OMIM) site. Correlationbetween the location of the gene encoding CJPDZ on a physicalchromosomal map and a specific disorder, or a predisposition to aspecific disorder, may help define the region of DNA associated withthat disorder. The nucleotide sequences of the invention may be used todetect differences in gene sequences among normal, carrier, and affectedindividuals.

[0172] In situ hybridization of chromosomal preparations and physicalmapping techniques, such as linkage analysis using establishedchromosomal markers, may be used for extending genetic maps. Often theplacement of a gene on the chromosome of another mammalian species, suchas mouse, may reveal associated markers even if the number or arm of aparticular human chromosome is not known. New sequences can be assignedto chromosomal arms by physical mapping. This provides valuableinformation to investigators searching for disease genes usingpositional cloning or other gene discovery techniques. Once the diseaseor syndrome has been crudely localized by genetic linkage to aparticular genomic region, e.g., ataxia-telangiectasia to 11q22-23, anysequences mapping to that area may represent associated or regulatorygenes for further investigation. (See, e.g., Gatti, R. A. et al. (1988)Nature 336:577-580.) The nucleotide sequence of the subject inventionmay also be used to detect differences in the chromosomal location dueto translocation, inversion, etc., among normal, carrier, or affectedindividuals.

[0173] In another embodiment of the invention, CJPDZ, its catalytic orimmunogenic fragments, or oligopeptides thereof can be used forscreening libraries of compounds in any of a variety of drug screeningtechniques. The fragment employed in such screening may be free insolution, affixed to a solid support, borne on a cell surface, orlocated intracellularly. The formation of binding complexes betweenCJPDZ and the agent being tested may be measured.

[0174] Another technique for drug screening provides for high throughputscreening of compounds having suitable binding affinity to the proteinof interest. (See, e.g., Geysen, et al. (1984) PCT applicationWO84/03564.) In this method, large numbers of different small testcompounds are synthesized on a solid substrate. The test compounds arereacted with CJPDZ, or fragments thereof, and washed. Bound CJPDZ isthen detected by methods well known in the art. Purified CJPDZ can alsobe coated directly onto plates for use in the aforementioned drugscreening techniques. Alternatively, non-neutralizing antibodies can beused to capture the peptide and immobilize it on a solid support.

[0175] In another embodiment, one may use competitive drug screeningassays in which neutralizing antibodies capable of binding CJPDZspecifically compete with a test compound for binding CJPDZ. In thismanner, antibodies can be used to detect the presence of any peptidewhich shares one or more antigenic determinants with CJPDZ.

[0176] In additional embodiments, the nucleotide sequences which encodeCJPDZ may be used in any molecular biology techniques that have yet tobe developed, provided the new techniques rely on properties ofnucleotide sequences that are currently known, including, but notlimited to, such properties as the triplet genetic code and specificbase pair interactions.

[0177] The examples below are provided to illustrate the subjectinvention and are not included for the purpose of limiting theinvention.

EXAMPLES

[0178] I. cDNA Library Construction

[0179] The UCMCL5T01 cDNA library was constructed using RNA isolatedfrom mononuclear cells obtained from umbilical cord blood of 12individuals. The cells were cultured for 12 days in the presence of IL-5prior to RNA isolation from the pooled lysates.

[0180] Cells were homogenized and lysed in guanidinium isothiocyanatesolution using a Brinkmann Homogenizer Polytron PT-3000 (BrinkmannInstruments, Westbury N.Y.). The lysate was centrifuged over a CsClcushion to isolate RNA. The RNA was extracted with acid phenol,precipitated with sodium acetate and ethanol, resuspended in RNase-freewater, and treated with DNase. Poly(A+) RNA was isolated using theOLIGOTEX mRNA purification kit (QIAGEN, Chatsworth Calif.).

[0181] Poly(A+) RNA was used for cDNA synthesis and construction of thecDNA library according to the recommended protocols in the SUPERSCRIPTplasmid system (Life Technologies). The cDNAs were fractionated on aSEPHAROSE CIAB column (Amersham Pharmacia Biotech), and those cDNAsexceeding 400 bp were ligated into pINCY (Incyte Genomics, Palo AltoCalif.). Recombinant plasmids were transformed into DH5α competent cells(Life Technologies).

[0182] II. Isolation of cDNA Clones

[0183] Plasmid DNA was released from the cells and purified using theREAL Prep 96 plasmid kit (QIAGEN). The recommended protocol was employedexcept for the following changes: 1) the bacteria were cultured in 1 mlof sterile Terrific Broth (Life Technologies) with carbenicillin at 25mg/l and glycerol at 0.4%; 2) after the cultures were incubated for 19hours, the cells were lysed with 0.3 ml of lysis buffer; and 3)following isopropanol precipitation, the plasmid DNA pellets were eachresuspended in 0.1 ml of distilled water. The DNA samples were stored at4° C.

[0184] III. Sequencing and Analysis

[0185] The cDNAs were prepared for sequencing using the ABI CATALYST 800(Perkin-Elmer) or the HYDRA microdispenser (Robbins Scientific) orMICROLAB 2200 (Hamilton) systems in combination with the PTC-200 thermalcyclers (MJ Research). The cDNAs were sequenced using the ABI PRISM 373or 377 sequencing systems (Perkin-Elmer) and standard ABI protocols,base calling software, and kits. In one alternative, cDNAs weresequenced using the MEGABACE 1000 DNA sequencing system (MolecularDynamics). In another alternative, the cDNAs were amplified andsequenced using the ABI PRISM BIGDYE Terminator cycle sequencing readyreaction kit (Perkin-Elmer). In yet another alternative, cDNAs weresequenced using solutions and dyes from Amersham Pharmacia Biotech.Reading frames for the ESTs were determined using standard methods(reviewed in Ausubel, 1997, supra, unit 7.7). Some of the cDNA sequenceswere selected for extension using the techniques disclosed in Example V.

[0186] The polynucleotide sequences derived from cDNA, extension, andshotgun sequencing were assembled and analyzed using a combination ofsoftware programs which utilize algorithms well known to those skilledin the art. Table 1 summarizes the software programs, descriptions,references, and threshold parameters used. The first column of Table 1shows the tools, programs, and algorithms used, the second columnprovides a brief description thereof, the third column presents thereferences which are incorporated by reference herein, and the fourthcolumn presents, where applicable, the scores, probability values, andother parameters used to evaluate the strength of a match between twosequences (the higher the probability the greater the homology).Sequences were analyzed using MACDNASIS PRO software (Hitachi SoftwareEngineering) and LASERGENE software (DNASTAR).

[0187] The polynucleotide sequences were validated by removing vector,linker, and polyA sequences and by masking ambiguous bases, usingalgorithms and programs based on BLAST, dynamic programming, anddinucleotide nearest neighbor analysis. The sequences were then queriedagainst a selection of public databases such as GenBank primate, rodent,mammalian, vertebrate, and eukaryote databases, and BLOCKS to acquireannotation, using programs based on BLAST, FASTA, and BLIMPS. Thesequences were assembled into full length polynucleotide sequences usingprograms based on Phred, Phrap, and Consed, and were screened for openreading frames using programs based on GeneMark, BLAST, and FASTA. Thefull length polynucleotide sequences were translated to derive thecorresponding full length amino acid sequences, and these full lengthsequences were subsequently analyzed by querying against databases suchas the GenBank databases (described above), SwissProt, BLOCKS, PRINTS,PFAM, and Prosite.

[0188] The programs described above for the assembly and analysis offull length polynucleotide and amino acid sequences were used toidentify polynucleotide sequence fragments from SEQ ID NO:2. Fragmentsfrom about 20 to about 4000 nucleotides which are useful inhybridization and amplification technologies were described in TheInvention section above.

[0189] IV. Northern Analysis

[0190] Northern analysis is a laboratory technique used to detect thepresence of a transcript of a gene and involves the hybridization of alabeled nucleotide sequence to a membrane on which RNAs from aparticular cell type or tissue have been bound. (See, e.g., Sambrook,supra, ch. 7; Ausubel, 1995, supra, ch. 4 and 16.)

[0191] Analogous computer techniques applying BLAST were used to searchfor identical or related molecules in nucleotide databases such asGenBank or LIFESEQ database (Incyte Genomics, Palo Alto Calif.). Thisanalysis is much faster than multiple membrane-based hybridizations. Inaddition, the sensitivity of the computer search can be modified todetermine whether any particular match is categorized as exact orsimilar. The basis of the search is the product score, which is definedas:$\frac{\% \quad {sequence}\quad {identity} \times \% \quad {maximum}\quad {BLAST}\quad {score}}{100}$

[0192] The product score takes into account both the degree ofsimilarity between two sequences and the length of the sequence match.For example, with a product score of 40, the match will be exact withina 1% to 2% error, and, with a product score of 70, the match will beexact. Similar molecules are usually identified by selecting those whichshow product scores between 15 and 40, although lower scores mayidentify related molecules.

[0193] The results of northern analyses are reported a percentagedistribution of libraries in which the transcript encoding CJPDZoccurred. Analysis involved the categorization of cDNA libraries byorgan/tissue and disease. The organ/tissue categories includedcardiovascular, dermatologic, developmental, endocrine,gastrointestinal, hematopoietic/immune, musculoskeletal, nervous,reproductive, and urologic. The disease categories included cancer,inflammation/trauma, fetal, neurological, and pooled. For each category,the number of libraries expressing the sequence of interest was countedand divided by the total number of libraries across all categories.Percentage values of tissue-specific and disease expression are reportedin the description of the invention.

[0194] V. Extension of CJPDZ Encoding Polynucleotides

[0195] The full length nucleic acid sequence of SEQ ID NO:2 was producedby extension of an appropriate fragment of the full length moleculeusing oligonucleotide primers designed from this fragment. One primerwas synthesized to initiate 5′ extension of the known fragment, and theother primer, to initiate 3′ extension of the known fragment. Theinitial primers were designed using OLIGO 4.06 software (NationalBiosciences), or another appropriate program, to be about 22 to 30nucleotides in length, to have a GC content of about 50% or more, and toanneal to the target sequence at temperatures of about 68° C. to about72° C. Any stretch of nucleotides which would result in hairpinstructures and primer-primer dimerizations was avoided.

[0196] Selected human cDNA libraries were used to extend the sequence.If more than one extension was necessary or desired, additional ornested sets of primers were designed.

[0197] High fidelity amplification was obtained by PCR using methodswell known in the art. PCR was performed in 96-well plates using thePTC-200 thermal cycler (MJ Research, Inc.). The reaction mix containedDNA template, 200 mmol of each primer, reaction buffer containing Mg²⁺,(NH₄)₂SO₄, and β-mercaptoethanol, Taq DNA polymerase (Amersham PharmaciaBiotech), ELONGASE enzyme (Life Technologies), and Pfu DNA polymerase(Stratagene), with the following parameters for primer pair PCI A andPCI B: Step 1: 94° C., 3 min; Step 2: 94° C., 15 sec; Step 3: 60° C., 1min; Step 4: 68° C., 2 min; Step 5: Steps 2, 3, and 4 repeated 20 times;Step 6: 68° C., 5 min; Step 7: storage at 4° C. In the alternative, theparameters for primer pair T7 and SK+ were as follows: Step 1: 94° C., 3min; Step 2: 94° C., 15 sec; Step 3: 57° C., 1 min; Step 4: 68° C., 2min; Step 5: Steps 2, 3, and 4 repeated 20 times; Step 6: 68° C., 5 min;Step 7: storage at 4° C.

[0198] The concentration of DNA in each well was determined bydispensing 100 μl PICO GREEN quantitation reagent (0.25% (v/v) PICOGREEN; Molecular Probes, Eugene Oreg.) dissolved in 1× TE and 0.5 μl ofundiluted PCR product into each well of an opaque fluorimeter plate(Corning Costar, Acton Mass.), allowing the DNA to bind to the reagent.The plate was scanned in a Fluoroskan II (Labsystems Oy, Helsinki,Finland) to measure the fluorescence of the sample and to quantify theconcentration of DNA. A 5 μl to 10 μl aliquot of the reaction mixturewas analyzed by electrophoresis on a 1% agarose mini-gel to determinewhich reactions were successful in extending the sequence.

[0199] The extended nucleotides were desalted and concentrated,transferred to 384-well plates, digested with CviJI cholera virusendonuclease (Molecular Biology Research, Madison Wis.), and sonicatedor sheared prior to religation into pUC 18 vector (Amersham PharmaciaBiotech). For shotgun sequencing, the digested nucleotides wereseparated on low concentration (0.6 to 0.8%) agarose gels, fragmentswere excised, and agar digested with Agar ACE (Promega). Extended cloneswere religated using T4 ligase (New England Biolabs, Beverly Mass.) intopUC 18 vector (Amersham Pharmacia Biotech), treated with Pfu DNApolymerase (Stratagene) to fill-in restriction site overhangs, andtransfected into competent E. coli cells. Transformed cells wereselected on antibiotic-containing media, individual colonies were pickedand cultured overnight at 37° C. in 384-well plates in LB/2x carb liquidmedia.

[0200] The cells were lysed, and DNA was amplified by PCR using Taq DNApolymerase (Amersham Pharmacia Biotech) and Pfu DNA polymerase(Stratagene) with the following parameters: Step 1: 94° C., 3 min; Step2: 94° C., 15 sec; Step 3: 60° C., 1 min; Step 4: 72° C., 2 min; Step 5:steps 2, 3, and 4 repeated 29 times; Step 6: 72° C., 5 min; Step 7:storage at 4° C. DNA was quantified by PICOGREEN reagent (MolecularProbes) as described above. Samples with low DNA recoveries werereamplified using the same conditions as described above. Samples werediluted with 20% dimethysulphoxide (1:2, v/v), and sequenced usingDYENAMIC energy transfer sequencing primers and the DYENAMIC DIRECT kit(Amersham Pharmacia Biotech) or the ABI PRISM BIGDYE Terminator cyclesequencing ready reaction kit (Perkin-Elmer).

[0201] In like manner, the nucleotide sequence of SEQ ID NO:2 is used toobtain 5′ regulatory sequences using the procedure above,oligonucleotides designed for such extension, and an appropriate genomiclibrary.

[0202] VI. Labeling and Use of Individual Hybridization Probes

[0203] Hybridization probes derived from SEQ ID NO:2 are employed toscreen cDNAs, genomic DNAs, or mRNAs. Although the labeling ofoligonucleotides, consisting of about 20 base pairs, is specificallydescribed, essentially the same procedure is used with larger nucleotidefragments. Oligonucleotides are designed using state-of-the-art softwaresuch as OLIGO 4.06 software (National Biosciences) and labeled bycombining 50 pmol of each oligomer, 250 μCi of [³²P]-adenosinetriphosphate (Amersham Pharmacia Biotech), and T4 polynucleotide kinase(DuPont NEN, Boston Mass.). The labeled oligonucleotides aresubstantially purified using a SEPHADEX G-25 superfine size exclusiondextran bead column (Amersham Pharmacia Biotech). An aliquot containing10⁷ counts per minute of the labeled probe is used in a typicalmembrane-based hybridization analysis of human genomic DNA digested withone of the following endonucleases: Ase I, Bgl II, Eco RI, Pst I, Xba I,or Pvu II (DuPont NEN).

[0204] The DNA from each digest is fractionated on a 0.7% agarose geland transferred to nylon membranes (Nytran Plus, Schleicher & Schuell,Durham N.H.). Hybridization is carried out for 16 hours at 40° C. Toremove nonspecific signals, blots are sequentially washed at roomtemperature under increasingly stringent conditions up to 0.1× salinesodium citrate and 0.5% sodium dodecyl sulfate. After XOMAT-AR film(Eastman Kodak, Rochester N.Y.) is exposed to the blots to film forseveral hours, hybridization patterns are compared visually.

[0205] VII. Microarrays

[0206] A chemical coupling procedure and an ink jet device can be usedto synthesize array elements on the surface of a substrate. (See, e.g.,Baldeschweiler, supra.) An array analogous to a dot or slot blot mayalso be used to arrange and link elements to the surface of a substrateusing thermal, UV, chemical, or mechanical bonding procedures. A typicalarray may be produced by hand or using available methods and machinesand contain any appropriate number of elements. After hybridization,nonhybridized probes are removed and a scanner used to determine thelevels and patterns of fluorescence. The degree of complementarity andthe relative abundance of each probe which hybridizes to an element onthe microarray may be assessed through analysis of the scanned images.

[0207] Full-length cDNAs, Expressed Sequence Tags (ESTs), or fragmentsthereof may comprise the elements of the microarray. Fragments suitablefor hybridization can be selected using software well known in the artsuch as LASERGENE software (DNASTAR). Full-length cDNAs, ESTs, orfragments thereof corresponding to one of the nucleotide sequences ofthe present invention, or selected at random from a cDNA libraryrelevant to the present invention, are arranged on an appropriatesubstrate, e.g., a glass slide. The cDNA is fixed to the slide using,e.g., UV cross-linking followed by thermal and chemical treatments andsubsequent drying. (See, e.g., Schena, M. et al. (1995) Science270:467-470; Shalon, D. et al. (1996) Genome Res. 6:639-645.)Fluorescent probes are prepared and used for hybridization to theelements on the substrate. The substrate is analyzed by proceduresdescribed above.

[0208] VIII. Complementary Polynucleotides

[0209] Sequences complementary to the CJPDZ-encoding sequences, or anyparts thereof, are used to detect, decrease, or inhibit expression ofnaturally occurring CJPDZ. Although use of oligonucleotides comprisingfrom about 15 to 30 base pairs is described, essentially the sameprocedure is used with smaller or with larger sequence fragments.Appropriate oligonucleotides are designed using OLIGO 4.06 software(National Biosciences) and the coding sequence of CJPDZ. To inhibittranscription, a complementary oligonucleotide is designed from the mostunique 5′ sequence and used to prevent promoter binding to the codingsequence. To inhibit translation, a complementary oligonucleotide isdesigned to prevent ribosomal binding to the CJPDZ-encoding transcript.

[0210] IX. Expression of CJPDZ

[0211] Expression and purification of CJPDZ are achieved using bacterialor virus-based expression systems. For expression of CJPDZ in bacteria,cDNA is subcloned into an appropriate vector containing an antibioticresistance gene and an inducible promoter that directs high levels ofcDNA transcription.

[0212] Examples of such promoters include, but are not limited to, thetrp-lac (tac) hybrid promoter and the T5 or T7 bacteriophage promoter inconjunction with the lac operator regulatory element. Recombinantvectors are transformed into suitable bacterial hosts, e.g., BL21(DE3).Antibiotic resistant bacteria express CJPDZ upon induction withisopropyl beta-D-thiogalactopyranoside (IPTG). Expression of CJPDZ ineukaryotic cells is achieved by infecting insect or mammalian cell lineswith recombinant Autographica californica nuclear polyhedrosis virus(AcMNPV), commonly known as baculovirus. The nonessential polyhedringene of baculovirus is replaced with cDNA encoding CJPDZ by eitherhomologous recombination or bacterial-mediated transposition involvingtransfer plasmid intermediates. Viral infectivity is maintained and thestrong polyhedrin promoter drives high levels of cDNA transcription.Recombinant baculovirus is used to infect Spodoptera frugiperda (Sf9)insect cells in most cases, or human hepatocytes, in some cases.Infection of the latter requires additional genetic modifications tobaculovirus. (See Engelhard, E. K. et al. (1994) Proc. Natl. Acad. Sci.USA 91:3224-3227; Sandig, V. et al. (1996) Hum. Gene Ther. 7:1937-1945.)

[0213] In most expression systems, CJPDZ is synthesized as a fusionprotein with, e.g., glutathione S-transferase (GST) or a peptide epitopetag, such as FLAG or 6-His, permitting rapid, single-step,affinity-based purification of recombinant fusion protein from crudecell lysates. GST, a 26-kilodalton enzyme from Schistosoma japonicum,enables the purification of fusion proteins on immobilized glutathioneunder conditions that maintain protein activity and antigenicity(Amersham Pharmacia Biotech). Following purification, the GST moiety canbe proteolytically cleaved from CJPDZ at specifically engineered sites.FLAG, an 8-amino acid peptide, enables immunoaffinity purification usingcommercially available monoclonal and polyclonal anti-FLAG antibodies(Eastman Kodak). 6-His, a stretch of six consecutive histidine residues,enables purification on metal-chelate resins (QIAGEN). Methods forprotein expression and purification are discussed in Ausubel (1995,supra, ch 10 and 16). Purified CJPDZ obtained by these methods can beused directly in the following activity assay.

[0214] X. Demonstration of CJPDZ Activity

[0215] An assay for CJPDZ activity measures the PDZ-induced clusteringof transmembrane receptors (Ponting, supra). Cultured cell lines arecotransfected with cDNA encoding CJPDZ and either EGF receptor or NMDAreceptor. Control cell lines are transfected with only one of the abovecDNAs. Clustering of EGF receptors or NMDA receptors in thecotransfected cell lines is detected and quantified using commerciallyavailable antibody specific to these receptors in conjunction withindirect immunofluorescence and image analysis systems. The amount ofreceptor clustering is directly proportional to the amount of CJPDZactivity.

[0216] XI. Functional Assays

[0217] CJPDZ function is assessed by expressing the sequences encodingCJPDZ at physiologically elevated levels in mammalian cell culturesystems. cDNA is subcloned into a mammalian expression vector containinga strong promoter that drives high levels of cDNA expression. Vectors ofchoice include pCMV SPORT (Life Technologies) and pCR3. 1 (Invitrogen,Carlsbad Calif.), both of which contain the cytomegalovirus promoter.5-10 μg of recombinant vector are transiently transfected into a humancell line, preferably of endothelial or hematopoietic origin, usingeither liposome formulations or electroporation. 1-2 μg of an additionalplasmid containing sequences encoding a marker protein arecotransfected. Expression of a marker protein provides a means todistinguish transfected cells from nontransfected cells and is areliable predictor of cDNA expression from the recombinant vector.Marker proteins of choice include, e.g., Green Fluorescent Protein (GFP;Clontech), CD64, or a CD64-GFP fusion protein. Flow cytometry (FCM), anautomated, laser optics-based technique, is used to identify transfectedcells expressing GFP or CD64-GFP, and to evaluate cellular properties,for example, their apoptotic state. FCM detects and quantifies theuptake of fluorescent molecules that diagnose events preceding orcoincident with cell death. These events include changes in nuclear DNAcontent as measured by staining of DNA with propidium iodide; changes incell size and granularity as measured by forward light scatter and 90degree side light scatter; down-regulation of DNA synthesis as measuredby decrease in bromodeoxyuridine uptake; alterations in expression ofcell surface and intracellular proteins as measured by reactivity withspecific antibodies; and alterations in plasma membrane composition asmeasured by the binding of fluorescein-conjugated Annexin V protein tothe cell surface. Methods in flow cytometry are discussed in Ormerod, M.G. (1994) Flow Cytometry, Oxford, New York N.Y.

[0218] The influence of CJPDZ on gene expression can be assessed usinghighly purified populations of cells transfected with sequences encodingCJPDZ and either CD64 or CD64-GFP. CD64 and CD64-GFP are expressed onthe surface of transfected cells and bind to conserved regions of humanimmunoglobulin G (IgG). Transfected cells are efficiently separated fromnontransfected cells using magnetic beads coated with either human IgGor antibody against CD64 (DYNAL, Lake Success N.Y.). mRNA can bepurified from the cells using methods well known by those of skill inthe art. Expression of mRNA encoding CJPDZ and other genes of interestcan be analyzed by northern analysis or microarray techniques.

[0219] XII. Production of CJPDZ Specific Antibodies

[0220] CJPDZ substantially purified using polyacrylamide gelelectrophoresis (PAGE; see, e.g., Harrington, M. G. (1990) MethodsEnzymol. 182:488-495), or other purification techniques, is used toimmunize rabbits and to produce antibodies using standard protocols.

[0221] Alternatively, the CJPDZ amino acid sequence is analyzed usingLASERGENE software (DNASTAR) to determine regions of highimmunogenicity, and a corresponding oligopeptide is synthesized and usedto raise antibodies by means known to those of skill in the art. Methodsfor selection of appropriate epitopes, such as those near the C-terminusor in hydrophilic regions are well described in the art. (See, e.g.,Ausubel, 1995, supra, ch. 11.)

[0222] Typically, oligopeptides 15 residues in length are synthesizedusing an ABI 431A Peptide Synthesizer (Perkin-Elmer) usingfmoc-chemistry and coupled to KLH (Sigma-Aldrich, St. Louis Mo.) byreaction with N-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) toincrease immunogenicity. (See, e.g., Ausubel, 1995, supra.) Rabbits areimmunized with the oligopeptide-KLH complex in complete Freund'sadjuvant. Resulting antisera are tested for antipeptide activity by, forexample, binding the peptide to plastic, blocking with 1% BSA, reactingwith rabbit antisera, washing, and reacting with radio-iodinated goatanti-rabbit IgG.

[0223] XIII. Purification of Naturally Occurring CJPDZ Using SpecificAntibodies

[0224] Naturally occurring or recombinant CJPDZ is substantiallypurified by immunoaffinity chromatography using antibodies specific forCJPDZ. An immunoaffinity column is constructed by covalently couplinganti-CJPDZ antibody to an activated chromatographic resin, such asCNBr-activated SEPHAROSE (Amersham Pharmacia Biotech). After thecoupling, the resin is blocked and washed according to themanufacturer's instructions.

[0225] Media containing CJPDZ are passed over the immunoaffinity column,and the column is washed under conditions that allow the preferentialabsorbance of CJPDZ (e.g., high ionic strength buffers in the presenceof detergent). The column is eluted under conditions that disruptantibody/CJPDZ binding (e.g., a buffer of pH 2 to pH 3, or a highconcentration of a chaotrope, such as urea or thiocyanate ion), andCJPDZ is collected.

[0226] XIV. Identification of Molecules Which Interact with CJPDZ

[0227] CJPDZ, or biologically active fragments thereof, are labeled with1251 Bolton-Hunter reagent. (See, e.g., Bolton et al. (1973) Biochem. J.133:529-539.) Candidate molecules previously arrayed in the wells of amulti-well plate are incubated with the labeled CJPDZ, washed, and anywells with labeled CJPDZ complex are assayed. Data obtained usingdifferent concentrations of CJPDZ are used to calculate values for thenumber, affinity, and association of CJPDZ with the candidate molecules.Various modifications and variations of the described methods andsystems of the invention will be apparent to those skilled in the artwithout departing from the scope and spirit of the invention. Althoughthe invention has been described in connection with specific preferredembodiments, it should be understood that the invention as claimedshould not be unduly limited to such specific embodiments. Indeed,various modifications of the described modes for carrying out theinvention which are obvious to those skilled in molecular biology orrelated fields are intended to be within the scope of the followingclaims. TABLE 1 Program Description Reference Parameter Threshold ABIFACTURA A program that removes Perkin-Elmer Applied Biosystems, vectorsequences and Foster City, CA. masks ambiguous bases in nucleic acidsequences. ABI/PARACEL FDF A Fast Data Finder useful Perkin-ElmerApplied Biosystems Mismatch <50% in comparing and annotating FosterCity, CA; Paracel Inc., Pasadena, CA. amino acid or nucleic acidsequences. ABI AutoAssembler A program that assembles Perkin-ElmerApplied Biosystems, nucleic acid sequences. Foster City, CA. BLAST ABasic Local Alignment Altschul, S. F. et al. (1990) J. Mol. Biol ETSs:Probability value = 1.0E−8 Search Tool useful in 215:403-410; Altschul,S. F. et al. (1997) or less sequence similarity search Nucleic AcidsRes, 25:3389-3402 Full Length sequences: Probability for amino acid andnucleic value = 1.0E−10 or less acid sequences. BLAST includes, fivefunctions: blastp, blasta, blastx, tblastn, and tblastx. FASTA A Pearsonand Lipman Pearson, W. R. and D. J. Lipman (1988) Proc. ESTs: fasta Evalue = 1.06E−6 algorithm that searches for Natl. Acad Sci.85:2444-2448; Pearson, W. R. Assembled ESTs: fasta Indentity =similarity between a query (1990) Methods Enzymol. 183:63-98; and 95% orgreater and Match sequence and a group of Smith, T. F. and M. S.Waterman (1981) Adv. length = 200 bases or greater; fastx sequences ofthe same type. Appl. Math. 2:482-489. E value = 1.0E−8 or less FASTAcomprises as least Full Length sequences: fastx five functions: fasta,score = 100 or greater tfasta, fastx, and ssearch. BLIMPS A BLocksIMProved Henikoff, S and J. G. Henikoff, Nucl. Acid Res., Score = 1000or greater; Ratio of Searcher that matches a 19:6565-72, 1991. J. G.Henikoff and S. Score/Strength = 0.75 or larger; sequence against thosein Henikoff (1996) Methods Enzymol. 266:88-105; and Probability value =1.0E−3 or BLOCKS and PRINTS and Attwood, T. K. et al. (1997) J. Chem.Inf. less databases to search for Comput. Sci. 37:417-424 gene families,sequence homology, and structural fingerprint regions. PFAM A HiddenMarkov Models- Krogh, A. et al. (1994) J. Mol. Biol., 235:1501- Score =10-50 bits, depending on based application 1531; Sonhammer, E. L. L. etal. (1988) individual protein families useful for protein Nucleic AcidsRes. 26:320-322. family search. ProfileScan An algorithm that searchesGribskov, M. et al. (1988) CABIOS 4:61-66; Score = 4.0 or greater forstructural and Gribskov, et al. (1989) Methods Enzymol. sequence motifsin protein 183:146-159; Bairoch, A. et al. (1997) Nucleic sequences thatmatch Acids Res. 25:217-221. sequence patterns defined in Prosite. PhredA base-calling algorithm Ewing, B. et al. (1998) Genome that examinesautomated Res. 8:175-185; Ewing, B. and P. sequencer traces with highGreen (1998) Genome Res. 8:186- sensitivity and probability. 194. PhrapA Phils Revised Assembly Smith, T. F. and M. S. Waterman (1981) Adv.Score = 120 or greater; Match Program including SWAT Appl. Math.2:482-489; Smith, T. F. and M. S. length = 56 or greater and CrossMatch,programs Waterman (1981) J. Mol. Biol. 147:195-197; based on efficientimple- and Green, P., University of Washington, mentation of the Smith-Seattle, WA. Waterman algorithm, useful in searching sequence homologyand assembling DNA sequences. Consed A graphical tool for Gordon, D. etal. (1998) Genome viewing and editing Phrap Res. 8:195-202. assembliesSPScan A weight matrix analysis Nielson, H. et al. (1997) ProteinEngineering Score = 5 or greater program that scans protein 10:1-6;Claverie, J. M. and S. Audic (1997) sequences for the presence CABIOS12:431-439. of secretory signal peptides. Motifs A program that searchesBairoch et al. supra; Wisconsin amino acid sequences for Package ProgramManual, version patterns that matched 9, page M51-59, Genetics Computerthose defined in Prosite. Group, Madison, WI.

[0228]

1 3 1 233 PRT Homo sapiens 1974337 1 Met Leu Lys Pro Ser Val Thr Ser AlaPro Thr Ala Asp Met Ala 1 5 10 15 Thr Leu Thr Val Val Gln Pro Leu ThrLeu Asp Arg Asp Val Ala 20 25 30 Arg Ala Ile Glu Leu Leu Glu Lys Leu GlnGlu Ser Gly Glu Val 35 40 45 Pro Val His Lys Leu Gln Ser Leu Lys Lys ValLeu Gln Ser Glu 50 55 60 Phe Cys Thr Ala Ile Arg Glu Val Tyr Gln Tyr MetHis Glu Thr 65 70 75 Ile Thr Val Asn Gly Cys Pro Glu Phe Arg Ala Arg AlaThr Ala 80 85 90 Lys Ala Thr Val Ala Ala Phe Ala Ala Ser Glu Gly His SerHis 95 100 105 Pro Arg Val Val Glu Leu Pro Lys Thr Asp Glu Gly Leu GlyPhe 110 115 120 Asn Val Met Gly Gly Lys Glu Gln Asn Ser Pro Ile Tyr IleSer 125 130 135 Arg Ile Ile Pro Gly Gly Val Ala Glu Arg His Gly Gly LeuLys 140 145 150 Arg Gly Asp Gln Leu Leu Ser Val Asn Gly Val Ser Val GluGly 155 160 165 Glu His His Glu Lys Ala Val Glu Leu Leu Lys Ala Ala LysAsp 170 175 180 Ser Val Lys Leu Val Val Arg Tyr Thr Pro Lys Val Leu GluGlu 185 190 195 Met Glu Ala Arg Phe Glu Lys Leu Arg Thr Ala Arg Arg ArgGln 200 205 210 Gln Gln Gln Leu Leu Ile Gln Gln Gln Gln Gln Gln Gln GlnGln 215 220 225 Gln Thr Gln Gln Asn His Met Ser 230 2 1396 DNA Homosapiens 1974337 2 cacgcacccg catgcacaca cgtatattct gaccatttta ttagagtggaaagttgaaag 60 gaagcaaccc gccagctaca cccacccagc gctcctgggg gtggaatagcaaagttctag 120 ggcagagcct tccctcccag agcccggcga tgcagtcgct ctcggatacctgctcagctc 180 cgcaccgcaa ctgaagatct gccgccgcgg aacagttgcg tctccatctggctaccaacc 240 cacccaagct ttcttctcca ccaccaccac cttccttcct tccccctcctccccctcctt 300 tcggtcctcc ctctccaccc ccgcccccaa tctcctcctt tttttctcactacgagcggt 360 tgctgatgct gaagccgagc gtcacttcgg ctcccacggc agacatggcgacattgacag 420 tggtccagcc gctcaccctg gacagagatg ttgcaagagc aattgaattactggaaaaac 480 tacaggaatc tggagaagta ccagtgcaca agctacaatc cctcaaaaaagtgcttcaga 540 gtgagttttg tacagctatt cgagaggtgt atcaatatat gcatgaaacgataactgtta 600 atggctgtcc cgaattccgt gcgagggcaa cagcaaaggc aacagttgcagcttttgcag 660 ctagtgaagg ccactcccac cctcgagtag ttgaactgcc aaagactgatgaaggccttg 720 gttttaatgt gatgggagga aaggagcaaa attcccccat ttatatctctcgcataattc 780 ctggaggggt ggctgaaaga cacggaggcc tcaaaagagg agaccagctgctatcagtga 840 acggagtgag tgtggaagga gaacaccatg agaaagctgt ggaactactcaaggctgcta 900 aagacagcgt caagctggtg gtgcgataca ccccaaaagt tctggaagaaatggaggctc 960 gctttgaaaa gctacgaaca gccaggcgtc ggcagcagca gcaattgctaattcagcagc 1020 agcaacagca gcagcagcaa caaacacaac aaaaccacat gtcataggcccttgagggaa 1080 agctacttga tcaaacatcc gatagtcaca aatttgaaac cgtgcttcagaatcccagca 1140 catagtaaaa gacaacactg ataattatac ctgtcaagaa gctgtgaacacatggtgtat 1200 aaattcttta ccaaggcaac tcaacacctt ctttctctgg gcttgaaccgccactgctca 1260 cgtgggcttt acatacattg accttccatt cactgcagtg ggaattctcagtgtgcagag 1320 ggagaggttt tctagtctgc aaactgaaac agtgtaagaa gaataaagtctatgactttt 1380 aaataaaaaa aaaaaa 1396 3 297 PRT Caenorhabditis elegansg1685067 3 Met Gly Leu Lys Gly Phe Thr Gly Ser Phe Gln Gln Ile Arg Gly 15 10 15 Leu Leu Arg Pro Pro Lys Asn Leu Pro Phe Arg Gly Ile Phe Arg 2025 30 Lys Asp Gly Glu Val Val Arg Lys Asp Asp Leu Leu Val Asn Gln 35 4045 Phe Lys Met Asn Tyr His Pro Gly Leu Asn Val Tyr Tyr Glu Asn 50 55 60Asp Arg Gly Glu Arg Leu Leu Arg Ala His Cys Asp Gly Ile Val 65 70 75 ArgIle Ser Gln Glu Lys Cys Asp Pro Asp Tyr Glu Ile Glu Glu 80 85 90 Met LysGly Tyr Glu Tyr Arg Lys Asp Val Asp Leu Tyr Lys Met 95 100 105 Thr PheAsn Met Asp Asn Pro Asp Gly Pro Asn Leu Glu Arg Asp 110 115 120 Val GlnArg Ile Leu Glu Leu Met Glu His Val Gln Lys Thr Gly 125 130 135 Glu ValAsn Asn Ala Lys Leu Ala Ser Leu Gln Gln Val Leu Gln 140 145 150 Ser GluPhe Phe Gly Ala Val Arg Glu Val Tyr Glu Thr Val Tyr 155 160 165 Glu SerIle Asp Ala Asp Thr Thr Pro Glu Ile Lys Ala Ala Ala 170 175 180 Thr AlaLys Ala Thr Val Ala Ala Phe Ala Ala Ala Glu Gly His 185 190 195 Ala HisPro Arg Ile Val Glu Leu Pro Lys Thr Asp Gln Gly Leu 200 205 210 Gly PheAsn Val Met Gly Gly Lys Glu Gln Asn Ser Pro Ile Tyr 215 220 225 Ile SerArg Ile Ile Pro Gly Gly Val Ala Asp Arg His Gly Gly 230 235 240 Leu LysArg Gly Asp Gln Leu Ile Ala Val Asn Gly Asn Val Glu 245 250 255 Ala GluCys His Glu Lys Ala Val Asp Leu Leu Lys Ser Ala Val 260 265 270 Gly SerVal Lys Leu Val Ile Arg Tyr Met Pro Lys Leu Leu Asp 275 280 285 Glu MetGlu Arg Arg Phe Glu Arg Gln Arg Ile Pro 290 295

What is claimed is:
 1. An isolated polypeptide selected from the groupconsisting of: a) a polypeptide comprising an amino acid sequence of SEQID NO: 1, b) a naturally occurring polypeptide comprising an amino acidsequence at least 90% identical to an amino acid sequence of SEQ ID NO:1, c) a biologically active fragment of a polypeptide having an aminoacid sequence of SEQ ID NO: 1, and d) an immunogenic fragment of apolypeptide having an amino acid sequence of SEQ ID NO:
 1. 2. Anisolated polypeptide of claim 1 comprising the amino acid sequence ofSEQ ID NO:
 1. 3. An isolated polynucleotide encoding a polypeptide ofclaim
 1. 4. An isolated polynucleotide encoding a polypeptide of claim2.
 5. An isolated polynucleotide of claim 4 comprising the sequence ofSEQ ID NO:2.
 6. A recombinant polynucleotide comprising a promotersequence operably linked to a polynucleotide of claim
 3. 7. A celltransformed with a recombinant polynucleotide of claim
 6. 8. Atransgenic organism comprising a recombinant polynucleotide of claim 6.9. A method for producing a polypeptide of claim 1, the methodcomprising: a) culturing a cell under conditions suitable for expressionof the polypeptide, wherein said cell is transformed with a recombinantpolynucleotide, and said recombinant polynucleotide comprises a promotersequence operably linked to a polynucleotide encoding the polypeptide ofclaim 1, and b) recovering the polypeptide so expressed.
 10. An isolatedantibody which specifically binds to a polypeptide of claim
 1. 11. Anisolated polynucleotide selected from the group consisting of: a) apolynucleotide comprising a polynucleotide sequence of SEQ ID NO:2, b) anaturally occurring polynucleotide comprising a polynucleotide sequenceat least 90% identical to a polynucleotide of SEQ ID NO:2, c) apolynucleotide complementary to a polynucleotide of a), d) apolynucleotide complementary to a polynucleotide of b), and e) an RNAequivalent of a)-d).
 12. An isolated polynucleotide comprising at least60 contiguous nucleotides of a polynucleotide of claim
 11. 13. A methodfor detecting a target polynucleotide in a sample, said targetpolynucleotide having a sequence of a polynucleotide of claim 11, themethod comprising: a) hybridizing the sample with a probe comprising atleast 20 contiguous nucleotides comprising a sequence complementary tosaid target polynucleotide in the sample, and which probe specificallyhybridizes to said target polynucleotide, under conditions whereby ahybridization complex is formed between said probe and said targetpolynucleotide or fragments thereof, and b) detecting the presence orabsence of said hybridization complex, and, optionally, if present, theamount thereof.
 14. A method of claim 13, wherein the probe comprises atleast 60 contiguous nucleotides.
 15. A method for detecting a targetpolynucleotide in a sample, said target polynucleotide having a sequenceof a polynucleotide of claim 11, the method comprising: a) amplifyingsaid target polynucleotide or fragment thereof using polymerase chainreaction amplification, and b) detecting the presence or absence of saidamplified target polynucleotide or fragment thereof, and, optionally, ifpresent, the amount thereof.
 16. A composition comprising a polypeptideof claim 1 and a pharmaceutically acceptable excipient.
 17. Acomposition of claim 16, wherein the polypeptide has an amino acidsequence of SEQ ID NO:2.
 18. A method for treating a disease orcondition associated with decreased expression of functional CJPDZ,comprising administering to a patient in need of such treatment thecomposition of claim
 16. 19. A method for screening a compound foreffectiveness as an agonist of a polypeptide of claim 1, the methodcomprising: a) exposing a sample comprising a polypeptide of claim 1 toa compound, and b) detecting agonist activity in the sample.
 20. Acomposition comprising an agonist compound identified by a method ofclaim 19 and a pharmaceutically acceptable excipient.
 21. A method fortreating a disease or condition associated with decreased expression offunctional CJPDZ, comprising administering to a patient in need of suchtreatment a composition of claim
 20. 22. A method for screening acompound for effectiveness as an antagonist of a polypeptide of claim 1,the method comprising: a) exposing a sample comprising a polypeptide ofclaim 1 to a compound, and b) detecting antagonist activity in thesample.
 23. A composition comprising an antagonist compound identifiedby a method of claim 22 and a pharmaceutically acceptable excipient. 24.A method for treating a disease or condition associated withoverexpression of functional CJPDZ, comprising administering to apatient in need of such treatment a composition of claim
 23. 25. Amethod of screening for a compound that specifically binds to thepolypeptide of claim 1, said method comprising the steps of: a)combining the polypeptide of claim 1 with at least one test compoundunder suitable conditions, and b) detecting binding of the polypeptideof claim 1 to the test compound, thereby identifying a compound thatspecifically binds to the polypeptide of claim
 1. 26. A method ofscreening for a compound that modulates the activity of the polypeptideof claim 1, said method comprising: a) combining the polypeptide ofclaim 1 with at least one test compound under conditions permissive forthe activity of the polypeptide of claim 1, b) assessing the activity ofthe polypeptide of claim 1 in the presence of the test compound, and c)comparing the activity of the polypeptide of claim 1 in the presence ofthe test compound with the activity of the polypeptide of claim 1 in theabsence of the test compound, wherein a change in the activity of thepolypeptide of claim 1 in the presence of the test compound isindicative of a compound that modulates the activity of the polypeptideof claim
 1. 27. A method for screening a compound for effectiveness inaltering expression of a target polynucleotide, wherein said targetpolynucleotide comprises a sequence of claim 5, the method comprising:a) exposing a sample comprising the target polynucleotide to a compound,under conditions suitable for the expression of the targetpolynucleotide, b) detecting altered expression of the targetpolynucleotide, and c) comparing the expression of the targetpolynucleotide in the presence of varying amounts of the compound and inthe absence of the compound.
 28. A method for assessing toxicity of atest compound, said method comprising: a) treating a biological samplecontaining nucleic acids with the test compound; b) hybridizing thenucleic acids of the treated biological sample with a probe comprisingat least 20 contiguous nucleotides of a polynucleotide of claim 11 underconditions whereby a specific hybridization complex is formed betweensaid probe and a target polynucleotide in the biological sample, saidtarget polynucleotide comprising a polynucleotide sequence of apolynucleotide of claim 11 or fragment thereof; c) quantifying theamount of hybridization complex; and d) comparing the amount ofhybridization complex in the treated biological sample with the amountof hybridization complex in an untreated biological sample, wherein adifference in the amount of hybridization complex in the treatedbiological sample is indicative of toxicity of the test compound.
 29. Adiagnostic test for a condition or disease associated with theexpression of CJPDZ in a biological sample comprising the steps of: a)combining the biological sample with an antibody of claim 10, underconditions suitable for the antibody to bind the polypeptide and form anantibody:polypeptide complex; and b) detecting the complex, wherein thepresence of the complex correlates with the presence of the polypeptidein the biological sample.
 30. The antibody of claim 10, wherein theantibody is: a) a chimeric antibody, b) a single chain antibody, c) aFab fragment, d) a F(ab′)2 fragment, or e) a humanized antibody.
 31. Acomposition comprising an antibody of claim 10 and an acceptableexcipient.
 32. A method of diagnosing a condition or disease associatedwith the expression of CJPDZ in a subject, comprising administering tosaid subject an effective amount of the composition of claim
 31. 33. Acomposition of claim 31, wherein the antibody is labeled.
 34. A methodof diagnosing a condition or disease associated with the expression ofCJPDZ in a subject, comprising administering to said subject aneffective amount of the composition of claim
 33. 35. A method ofpreparing a polyclonal antibody with the specificity of the antibody ofclaim 10 comprising: a) immunizing an animal with a polypeptide havingan amino acid sequence of SEQ ID NO: 1, or an immunogenic fragmentthereof, under conditions to elicit an antibody response; b) isolatingantibodies from said animal; and c) screening the isolated antibodieswith the polypeptide, thereby identifying a polyclonal antibody whichbinds specifically to a polypeptide having an amino acid sequence of SEQID NO:
 1. 36. An antibody produced by a method of claim
 35. 37. Acomposition comprising the antibody of claim 36 and a suitable carrier.38. A method of making a monoclonal antibody with the specificity of theantibody of claim 10 comprising: a) immunizing an animal with apolypeptide having an amino acid sequence of SEQ ID NO: 1, or animmunogenic fragment thereof, under conditions to elicit an antibodyresponse; b) isolating antibody producing cells from the animal; c)fusing the antibody producing cells with immortalized cells to formmonoclonal antibody-producing hybridoma cells; d) culturing thehybridoma cells; and e) isolating from the culture monoclonal antibodywhich binds specifically to a polypeptide having an amino acid sequenceof SEQ ID NO:
 1. 39. A monoclonal antibody produced by a method of claim38.
 40. A composition comprising the antibody of claim 39 and a suitablecarrier.
 41. The antibody of claim 10, wherein the antibody is producedby screening a Fab expression library.
 42. The antibody of claim 10,wherein the antibody is produced by screening a recombinantimmunoglobulin library.
 43. A method for detecting a polypeptide havingan amino acid sequence of SEQ ID NO: 1 in a sample, comprising the stepsof: a) incubating the antibody of claim 10 with a sample underconditions to allow specific binding of the antibody and thepolypeptide; and b) detecting specific binding, wherein specific bindingindicates the presence of a polypeptide having an amino acid sequence ofSEQ ID NO: 1 in the sample.
 44. A method of purifying a polypeptidehaving an amino acid sequence of SEQ ID NO: 1 from a sample, the methodcomprising: a) incubating the antibody of claim 10 with a sample underconditions to allow specific binding of the antibody and thepolypeptide; and b) separating the antibody from the sample andobtaining the purified polypeptide having an amino acid sequence of SEQID NO:
 1. 45. An isolated antibody which specifically binds apolypeptide of claim 2.